A Method for Next-Generation Sequencing of Paired Diagnostic and Remission Samples to Detect Mitochondrial DNA Mutations Associated with Leukemia Ilaria.

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Presentation transcript:

A Method for Next-Generation Sequencing of Paired Diagnostic and Remission Samples to Detect Mitochondrial DNA Mutations Associated with Leukemia Ilaria S. Pagani, Chung H. Kok, Verity A. Saunders, Mark B. Van der Hoek, Susan L. Heatley, Anthony P. Schwarer, Christopher N. Hahn, Timothy P. Hughes, Deborah L. White, David M. Ross The Journal of Molecular Diagnostics Volume 19, Issue 5, Pages 711-721 (September 2017) DOI: 10.1016/j.jmoldx.2017.05.009 Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic representation of the pipeline for mutation calling. For each test [eg, diagnosis (Dx)] and its matched control (eg, remission) sample, LoFreq software was used to identify somatic mutations in the test sample based on comparison with the revised Cambridge Reference Sequence (rCRS). Variants that were present in both test and matched control samples were filtered out, and an unfiltered list of putative somatic mutations was generated. Subsequently, an empirically determined variant allele fraction threshold of >2% was applied to reduce the risk of false discovery because of sequencing errors, leaving the final list of filtered putative somatic mutations in the test sample. SNV, single-nucleotide variant. The Journal of Molecular Diagnostics 2017 19, 711-721DOI: (10.1016/j.jmoldx.2017.05.009) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Empirical determination of a threshold variant allele fraction (VAF) to distinguish true positives from sequencing errors. Two independent MiSeq runs were compared to assess the performance of the method. Cutoffs in the range of 1% to 5% were applied. A: Sensitivity (x axis) is plotted against specificity (y axis) for each cutoff. B: Positive predictive value (y axis) was calculated for each cutoff (x axis) and shows little increase above a cutoff of 2%. C: The technical error (VAF %; log10 scale) calculated for each single-nucleotide variant (y axis) was plotted against its position in the mitochondrial DNA (mtDNA) genome [revised Cambridge Reference Sequence (rCRS)] (x axis). The Journal of Molecular Diagnostics 2017 19, 711-721DOI: (10.1016/j.jmoldx.2017.05.009) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Putative somatic mutations identified in remission blood samples. A: The variant allele fractions (VAFs; %) of the somatic mutations identified at diagnosis (Dx) were plotted against the VAFs (%) of the somatic mutations identified in remission using a nonhematopoietic tissue as control. B and C: VAF (%) of mitochondrial DNA (mtDNA) mutations (gray dots) in comparison with the BCR-ABL1IS ratio (black squares) in two patients at Dx and in remission samples (log10 scale). Dotted lines indicate the threshold for detection of a somatic mutation. D: Sixteen new heteroplasmic mtDNA mutations were identified in remission samples in 12 of 26 (46%) patients. Their VAF (%; x axis) was higher than the corresponding BCR-ABL1IS mRNA levels (log10 scale; y axis). FU, follow-up; HF, hair follicle; MSC, mesenchymal stromal cell; Undet, undetectable. The Journal of Molecular Diagnostics 2017 19, 711-721DOI: (10.1016/j.jmoldx.2017.05.009) Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions