ELN 2021 molecular MRD recommendations based on a Delphi poll
| No. . | Molecular MRD recommendation . | LoE . | GoR . | LoA (%) . |
|---|---|---|---|---|
| B1 | Techniques for molecular MRD assessment should reach an LOD of 10−3 or lower. To achieve this LOD, qPCR, dPCR, or error-corrected NGS using UMIs is recommended. | IV | B | 100 |
| B2 | Either EDTA or heparin can be used on samples as an anticoagulant for molecular MRD analysis. | V | C | 76 |
| B3 | Only 5 mL of BM aspirate should be used for molecular MRD assessment from the first pull (or the first pull after repositioning, if the initial pull is used for flow-MRD). | V | B | 94 |
| B4 | The method of cell isolation should be kept consistent, as it may alter the leukemic cell percentage (eg, Ficoll separation to reduce dilution of leukemic cells with normal granulocytes or lysis of whole blood). | V | B | 82 |
| B5 | Leukemia-specific PCR assays (eg, for NPM1, PML-RARA, or CBF AML) are preferred over fewer specific markers, such as WT1 or EVI1 expression. | V | B | 78 |
| B6 | Targeted NGS-MRD using specific mutations identified at diagnosis vs agnostic panel approaches has different strengths and limitations, but both approaches can be considered, depending on sensitivity, turnaround time, resource use, setting (research, clinical trial, or clinical routine), and ability to standardize methodology and reporting. | IV | B | 88 |
| B7 | If a panel approach is used for NGS-MRD, emerging variants not found at diagnosis should be reported only if confidently detected above background noise. | IV | B | 89 |
| B8 | For NGS-MRD, we recommend considering all detected mutations as potential MRD markers, with the limitations detailed in recommendations B9 to B11. | IV | B | 100 |
| B9 | Germline mutations (VAF of ∼50 in genes ANKRD26, CEBPA, DDX41, ETV6, GATA2, RUNX1, and TP53) should be excluded as NGS-MRD markers, as they are noninformative for MRD. | V | A | 94 |
| B10 | Mutations in DNMT3A, TET2, and ASXL1 (DTA) can be found in age-related clonal hematopoiesis and should be excluded from MRD analysis. | IV | A | 100 |
| B11 | Mutations in signaling pathway genes (FLT3-ITD, FLT3-TKD, KIT, and RAS, among others) most likely represent residual AML when detected, but are often subclonal and have a low negative predictive value. These mutations are best used in combination with additional MRD markers. | IV | B | 94 |
| B12 | NGS-MRD analysis in patients treated with targeted agents (FLT3 inhibitors and IDH1/IDH2 inhibitors) should include the molecular marker that is targeted, but also others that are present in the sample. | V | A | 94 |
| B13 | As of this writing, there is no uniform bioinformatics pipeline/platform for NGS-MRD variant calling. Harmonization efforts are strongly recommended, preferably using published open-source algorithms. | V | A | 94 |
| B14 | Potential cross-sample sequence contamination as a result of pooling samples in NGS-MRD should be bioinformatically evaluated. | V | A | 100 |
| No. . | Molecular MRD recommendation . | LoE . | GoR . | LoA (%) . |
|---|---|---|---|---|
| B1 | Techniques for molecular MRD assessment should reach an LOD of 10−3 or lower. To achieve this LOD, qPCR, dPCR, or error-corrected NGS using UMIs is recommended. | IV | B | 100 |
| B2 | Either EDTA or heparin can be used on samples as an anticoagulant for molecular MRD analysis. | V | C | 76 |
| B3 | Only 5 mL of BM aspirate should be used for molecular MRD assessment from the first pull (or the first pull after repositioning, if the initial pull is used for flow-MRD). | V | B | 94 |
| B4 | The method of cell isolation should be kept consistent, as it may alter the leukemic cell percentage (eg, Ficoll separation to reduce dilution of leukemic cells with normal granulocytes or lysis of whole blood). | V | B | 82 |
| B5 | Leukemia-specific PCR assays (eg, for NPM1, PML-RARA, or CBF AML) are preferred over fewer specific markers, such as WT1 or EVI1 expression. | V | B | 78 |
| B6 | Targeted NGS-MRD using specific mutations identified at diagnosis vs agnostic panel approaches has different strengths and limitations, but both approaches can be considered, depending on sensitivity, turnaround time, resource use, setting (research, clinical trial, or clinical routine), and ability to standardize methodology and reporting. | IV | B | 88 |
| B7 | If a panel approach is used for NGS-MRD, emerging variants not found at diagnosis should be reported only if confidently detected above background noise. | IV | B | 89 |
| B8 | For NGS-MRD, we recommend considering all detected mutations as potential MRD markers, with the limitations detailed in recommendations B9 to B11. | IV | B | 100 |
| B9 | Germline mutations (VAF of ∼50 in genes ANKRD26, CEBPA, DDX41, ETV6, GATA2, RUNX1, and TP53) should be excluded as NGS-MRD markers, as they are noninformative for MRD. | V | A | 94 |
| B10 | Mutations in DNMT3A, TET2, and ASXL1 (DTA) can be found in age-related clonal hematopoiesis and should be excluded from MRD analysis. | IV | A | 100 |
| B11 | Mutations in signaling pathway genes (FLT3-ITD, FLT3-TKD, KIT, and RAS, among others) most likely represent residual AML when detected, but are often subclonal and have a low negative predictive value. These mutations are best used in combination with additional MRD markers. | IV | B | 94 |
| B12 | NGS-MRD analysis in patients treated with targeted agents (FLT3 inhibitors and IDH1/IDH2 inhibitors) should include the molecular marker that is targeted, but also others that are present in the sample. | V | A | 94 |
| B13 | As of this writing, there is no uniform bioinformatics pipeline/platform for NGS-MRD variant calling. Harmonization efforts are strongly recommended, preferably using published open-source algorithms. | V | A | 94 |
| B14 | Potential cross-sample sequence contamination as a result of pooling samples in NGS-MRD should be bioinformatically evaluated. | V | A | 100 |
GoR, grade of recommendation; LoA, level of agreement; LOD, limit of detection; LoE, level of evidence; UMI, unique molecular identifier.