Louise Pettersson1,2, Sofie Johansson Alm3, Alvar Almstedt4, Yilun Chen5, Gustav Orrsjö6, Giti Shah-Barkhordar7, Li Zhou8, Heike Kotarsky8, Karina Vidovic1, Julia Asp3,9, Vladimir Lazarevic10, Lao H Saal5,11, Linda Fogelstrand3,9, Mats Ehinger1,8. 1. Department of Clinical Sciences, Division of Pathology, Lund University, Skane University Hospital, Lund, Sweden. 2. Department of Pathology, Halland Hospital Halmstad, Region Halland, Halmstad, Sweden. 3. Department of Laboratory Medicine, Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden. 4. SciLife Clinical Genomics Gothenburg, Gothenburg, Sweden. 5. Department of Clinical Sciences, Division of Oncology, Faculty of Medicine, Lund University, Lund, Sweden. 6. Section for Hematology and Coagulation, Sahlgrenska University Hospital, Gothenburg, Sweden. 7. Department of Clinical Genetics and Genomics, Sahlgrenska University Hospital, Gothenburg, Sweden. 8. Klinisk Patologi, Region Laboratories, Region Skåne, Lund, Sweden. 9. Department of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden. 10. Department of Hematology, Oncology and Radiation Physics, Lund University, Skane University Hospital, Lund, Sweden. 11. Lund University Cancer Center, Medicon Village, Lund, Sweden.
Abstract
INTRODUCTION: Reverse transcriptase quantitative PCR (RT-qPCR) is considered the method of choice for measurable residual disease (MRD) assessment in NPM1-mutated acute myeloid leukemia (AML). MRD can also be determined with DNA-based methods offering certain advantages. We here compared the DNA-based methods quantitative PCR (qPCR), droplet digital PCR (ddPCR), and targeted deep sequencing (deep seq) with RT-qPCR. METHODS: Of 110 follow-up samples from 30 patients with NPM1-mutated AML were analyzed by qPCR, ddPCR, deep seq, and RT-qPCR. To select DNA MRD cutoffs for bone marrow, we performed receiver operating characteristic analyses for each DNA method using prognostically relevant RT-qPCR cutoffs. RESULTS: The DNA-based methods showed strong intermethod correlation, but were less sensitive than RT-qPCR. A bone marrow cutoff at 0.1% leukemic DNA for qPCR or 0.05% variant allele frequency for ddPCR and deep seq offered optimal sensitivity and specificity with respect to 3 log10 reduction of NPM1 transcripts and/or 2% mutant NPM1/ABL. With these cutoffs, MRD results agreed in 95% (191/201) of the analyses. Although more sensitive, RT-qPCR failed to detect leukemic signals in 10% of samples with detectable leukemic DNA. CONCLUSION: DNA-based MRD techniques may complement RT-qPCR for assessment of residual leukemia. DNA-based methods offer high positive and negative predictive values with respect to residual leukemic NPM1 transcripts at levels of importance for response to treatment. However, moving to DNA-based MRD methods will miss a proportion of patients with residual leukemic RNA, but on the other hand some MRD samples with detectable leukemic DNA can be devoid of measurable leukemic RNA.
INTRODUCTION: Reverse transcriptase quantitative PCR (RT-qPCR) is considered the method of choice for measurable residual disease (MRD) assessment in NPM1-mutated acute myeloid leukemia (AML). MRD can also be determined with DNA-based methods offering certain advantages. We here compared the DNA-based methods quantitative PCR (qPCR), droplet digital PCR (ddPCR), and targeted deep sequencing (deep seq) with RT-qPCR. METHODS: Of 110 follow-up samples from 30 patients with NPM1-mutated AML were analyzed by qPCR, ddPCR, deep seq, and RT-qPCR. To select DNA MRD cutoffs for bone marrow, we performed receiver operating characteristic analyses for each DNA method using prognostically relevant RT-qPCR cutoffs. RESULTS: The DNA-based methods showed strong intermethod correlation, but were less sensitive than RT-qPCR. A bone marrow cutoff at 0.1% leukemic DNA for qPCR or 0.05% variant allele frequency for ddPCR and deep seq offered optimal sensitivity and specificity with respect to 3 log10 reduction of NPM1 transcripts and/or 2% mutant NPM1/ABL. With these cutoffs, MRD results agreed in 95% (191/201) of the analyses. Although more sensitive, RT-qPCR failed to detect leukemic signals in 10% of samples with detectable leukemic DNA. CONCLUSION: DNA-based MRD techniques may complement RT-qPCR for assessment of residual leukemia. DNA-based methods offer high positive and negative predictive values with respect to residual leukemic NPM1 transcripts at levels of importance for response to treatment. However, moving to DNA-based MRD methods will miss a proportion of patients with residual leukemic RNA, but on the other hand some MRD samples with detectable leukemic DNA can be devoid of measurable leukemic RNA.