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Quantification of the Mutant CALR Allelic Burden by Digital PCR

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Presentation on theme: "Quantification of the Mutant CALR Allelic Burden by Digital PCR"— Presentation transcript:

1 Quantification of the Mutant CALR Allelic Burden by Digital PCR
Olivier Mansier, Marina Migeon, Arnaud Saint-Lézer, Chloé James, Emmanuelle Verger, Marie Robin, Gérard Socié, Audrey Bidet, François-Xavier Mahon, Bruno Cassinat, Eric Lippert  The Journal of Molecular Diagnostics  Volume 18, Issue 1, Pages (January 2016) DOI: /j.jmoldx Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2 Figure 1 Sensitivity and accuracy of fragment analysis for CALR allelic burden assessment. Granulocyte DNA from patients with 50% CALR mutation type 1 (A) and type 2 (B) was diluted into normal DNA. Allelic burden was determined by fragment analysis after a 35-cycle PCR performed on 75 ng of DNA. Theoretical allelic burden was calculated by dividing mean allelic burden assessed on pure sample by the dilution factor. The bottom panel focuses on low mutational loads. Each point represents a replicate of three analyses. The Journal of Molecular Diagnostics  , 68-74DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3 Figure 2 Sensitivity and accuracy of droplet digital PCR (ddPCR) for CALR allelic burden assessment. Granulocyte DNA from patients with 50% CALR mutation type 1 (A) and type 2 (B) was diluted into normal DNA. Allelic burden was determined by ddPCR using 75 ng of DNA. Theoretical allelic burden was calculated by dividing mean allelic burden assessed on pure sample by the dilution factor. The bottom panel focuses on low mutational loads. Each point represents a replicate of three analyses. The Journal of Molecular Diagnostics  , 68-74DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4 Figure 3 Comparison of allelic burdens determined by fragment analysis and droplet digital PCR (ddPCR) on patients' samples. Allelic burdens of CALR mutations were assessed by fragment analysis and ddPCR. A: The results obtained by the two techniques using primary patients' samples (P < 0.0001, Wilcoxon matched-pairs signed rank test). Bland and Altman representation reveals the bias linked to fragment analysis compared to ddPCR with primary patients' samples (B) or DNA dilutions for type 1 (C) and type 2 (D) mutants. The Journal of Molecular Diagnostics  , 68-74DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5 Figure 4 Reliability of allelic burden determined by fragment analysis and droplet digital PCR (ddPCR). Samples that contained known levels of mutant CALR were prepared by mixing plasmid DNAs with known copy numbers of mutant CALR and normal genomic DNA with known amounts of wild-type CALR, both independently determined by absolute quantification using a non–allele-specific Evagreen-based ddPCR. Allelic burden was assessed for each dilution with fragment analysis and ddPCR. The observed values (y axis) are compared with expected values (x axis) for type 1 or type 2 CALR mutations. Each point represents one of five (top panels) or two (bottom panels) independent experiments. The Journal of Molecular Diagnostics  , 68-74DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6 Figure 5 Fragment analysis accuracy in nonsaturating conditions. Samples that contained known levels of mutant CALR were prepared by mixing plasmid DNAs with known copy numbers of mutant CALR and normal genomic DNA with known amounts of wild-type CALR, both independently determined by absolute quantification using a non–allele-specific Evagreen-based ddPCR. Allelic burden was assessed for each dilution with fragment analysis after different PCR cycle numbers and different dilutions of the PCR product. The Journal of Molecular Diagnostics  , 68-74DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7 Figure 6 Use of droplet digital PCR (ddPCR) for monitoring residual disease in hematopoietic stem cell transplant (HSCT) patients. Droplet digital PCR was used to determine allelic burdens in four patients before and after HSCT for myelofibrosis. The vertical arrow indicates the HSCT. The insets represent an enlargement of the boxed area. The Journal of Molecular Diagnostics  , 68-74DOI: ( /j.jmoldx ) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

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