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Direct comparison of quantitative digital PCR and 2-hydroxyglutarate enantiomeric ratio for IDH mutant allele frequency assessment in myeloid malignancy

Leukemia volume 29, pages 2421–2423 (2015)Cite this article

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Mutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) occur in ~15–20% of patients with acute myeloid leukemia (AML).1 A consequence is the aberrant accumulation of the (d)-enantiomer of 2-hydroxyglutarate (d-2-HG), a putative oncometabolite that competitively inhibits α-ketoglutarate-dependent epigenetic regulators such as TET2 and Jumonji domain histone demethylases.2 The presence of elevated 2-HG (either total or more specifically the d-enantiomer) in plasma or serum accurately identifies AML patients with IDH1 or IDH2 mutations, and 2-HG levels mirror disease burden.3, 4, 5, 6 However, it is not yet known whether 2-HG measurement represents the optimal approach to monitoring IDH mutant (IDHmut) allele burden during and after therapy, or whether alternative strategies such as allele-specific digital PCR may be equivalent or superior. Indeed, an evaluation of digital PCR in IDH-mutated AML has not yet been reported. Accurate assessment of mutant allele burden is essential to guide therapy and to predict prognosis, the more so given recent promising results from early phase trials of selective IDHmut inhibitors.7 To address this question, we have developed an allele-specific digital PCR method for recurring IDH1 and IDH2 mutations in AML, and made a direct comparison with 2-HG d:l enantiomeric ratios in concomitantly collected plasma samples. The 2-HG d:l ratio has recently been reported to be a more specific test for detection of patients with IDH mutations than total 2-HG alone.6 Digital PCR enables the quantitative identification of single-nucleotide variants (SNVs) against a wild-type DNA background. It relies on the random distribution of DNA molecules in multiple partitions of starting sample. Provided a sufficient number of partitions, and sufficient dilution to ensure that not every partition contains a target, Poisson statistics can estimate the number of SNV targets per partition in a binary manner, independent of exponential kinetics or reliance on standard curves. It has been made clinically practicable by the advent of nanofluidic and droplet-based partitioning platforms and has been applied to quantitate rare mutations in several cancers, including NPM1mut AML8 and ABL kinase domain mutations in chronic myeloid leukemia.9 It is more sensitive than Sanger sequencing (which has a mutant allele frequency (MAF) detection threshold of ~5–20%), and less burdensome and substantially cheaper than next-generation sequencing.

We assessed the d-2-HG:l-2-HG ratio in plasma samples collected from 192 consecutive patients presenting between February 2010 and December 2014 to The Christie NHS Foundation Trust in Manchester, UK, with newly diagnosed refractory cytopenia with multilineage dysplasia (RCMD; n=21), refractory anemia with excess blasts (RAEB; n=28) or AML (n=116); or with relapsed AML (n=27; Supplementary Figure S1 and Supplementary Table S1). In each case platelet-poor plasma and mononuclear cells from blood and/or bone marrow (BM) were cryopreserved at presentation and at frequent intervals thereafter. IDHmut cases were identified by Sanger sequencing of genomic DNA from presentation samples, with the exception of two cases where only blood mononuclear cells containing a low frequency of blasts were available for analysis.5 In these cases digital PCR provided the diagnosis where Sanger sequencing was unable to. Presentation and follow-up plasma samples were blindly screened for 2-HG stereoisomers by ultra-performance liquid chromatography with tandem mass spectrometry (Supplementary Figure S2).10 For digital PCR, we first designed and validated Taqman SNP genotyping quantitative PCR assays for the six most common IDH mutations (IDH1R132C/G/H/S; IDH2R140Q/R172K), comprising pooled common primers with FAM- and VIC-dye-labeled probes specific for each mutant or wild-type sequence, respectively (Supplementary Figures S3–S5). Allelic discrimination quantitative PCR clearly and correctly distinguished IDHmut from IDHwt patients in all replicates from all cases for each assay (Supplementary Figure S5). We then used these validated quantitative PCR assays in a digital PCR protocol using a nanofluidic platform and 37 K integrated fluidic circuit chips (Fluidigm, San Francisco, CA, USA; Supplementary Information). Digital PCR assays provided excellent reproducibility between replicates throughout the MAF range (R2=0.994; Supplementary Figures S6A and B) and were validated as quantitative to ⩽0.1% in spiking experiments (Supplementary Figure S7).

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Acknowledgements

This work was supported by Cancer Research UK grant number C5759/A20971, and a Leukaemia and Lymphoma Research clinical training fellowship to DW.

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Authors and Affiliations

  1. Leukaemia Biology Laboratory, Cancer Research UK Manchester Institute, The University of Manchester, Manchester, UK

    D H Wiseman & T C P Somervaille

  2. Department of Clinical Chemistry, Metabolic Laboratory, Free University Medical Center, Amsterdam, The Netherlands

    E A Struys, E E W Jansen & G S Salomons

  3. Biobank, Manchester Cancer Research Centre, Cancer Research UK Manchester Institute, The University of Manchester, Manchester, UK

    D P Wilks

  4. Molecular Biology Core Facility, Cancer Research UK Manchester Institute, The University of Manchester, Manchester, UK

    C I Clark

  5. Department of Haematology, The Christie NHS Foundation Trust, Manchester, UK

    M W Dennis

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Wiseman, D., Struys, E., Wilks, D. et al. Direct comparison of quantitative digital PCR and 2-hydroxyglutarate enantiomeric ratio for IDH mutant allele frequency assessment in myeloid malignancy. Leukemia 29, 2421–2423 (2015). https://doi.org/10.1038/leu.2015.151

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