AML MRD a consensus document from the European LeukemiaNet MRD Working Party .pptx

-Minimal/Measurable Residual Disease in AML: a consensus document from the European LeukemiaNet MRD Working Party

1. Journal club -Minimal/Measurable Residual Disease in AML: a consensus document from the European LeukemiaNet MRD Working Party

2. Application of MRD in AML Provide an objective methodology to establish a deeper remission status Refine outcome prediction and inform postremission treatment Identify impending relapse and enable early intervention Allow more robust posttransplant surveillance Use as a surrogate end point to accelerate drug testing and approval. * Numerous studies have investigated the value of MRD in AML and have consistently shown that MRD negativity, as defined by specified cutoff values, is highly prognostic for outcome- Gerrit J. Schuurhuis , Michael Heuser, et.; Minimal/measurable residual disease in AML: a consensus document from the European LeukemiaNet MRD Working Party. Blood 2018; 131 (12): 1275–1291. doi : https://doi.org/10.1182/blood-2017-09-801498

3. Study Method 2018 - An international panel of 24 experts, including 19 from European countries and 5 from the United States, met 4 times during 2016 and 2017, with numerous e-mail exchanges during this time. The panel included members with recognized technical, clinical, and translational knowledge of MRD in AML, including specific expertise on MFC MRD, molecular MRD, NGS, and clinical issues. For the clinical section, only MRD publications including at least 50 patients were reviewed (Table 1). Unpublished technical details from individual laboratory directors were also discussed and used. 2021- New and revised recommendations were established during in-person and online meetings, and a 2-stage Delphi poll was conducted to optimize consensus. All recommendations are graded by levels of evidence and agreement.

4. Highly sensitive and considered gold standard however only applicable in 40% of AML cases

5. Molecular MRD-points Need suitable markers NPM1 , RUNX1-RUNX1T1 , CBFB- MYH11, PML-RARA, KMT2A-MLLT3 , DEK-NUP214 , BCR-ABL , and WT1 (less specific, if only available abnormality PB sample should be used as high expression of WT1 in normal marrow) . Germline mutations (variant allele frequency [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 DTA mutations (DNMTA3A, TET2, ASXL10) can be found in age-related clonal hematopoiesis and should be excluded from MRD analysis (recommendation B10), as mutations associated with clonal hematopoiesis often persist during remission and thus may not represent the leukemic clone

6. Molecular Response (2018 guidelines) Complete Molecular Response Complete morphological remission + 2 successive MRD negative samples obtained within an interval of ≥4 weeks at a sensitivity level of at least 1 in 1000. Negative MRD in the presence of blasts suggests molecular loss of the particular marker. Molecular persistence at low copy numbers associated with a low risk of relapse. MRD with low copy numbers in patients with morphological CR (<100-200 copies/10 4 ABL copies corresponding to <1% to 2% of target to reference gene or allele burden) 53,54  and a copy number or relative increase <1 log between any 2 positive samples collected after the end of treatment. Molecular progression We define molecular progression in patients with molecular persistence at low copy number as an increase of MRD copy numbers ≥1 log 10 between any 2 positive samples. Molecular relapse Patients in complete morphological remission who achieve molecular remission may convert to positive MRD. We define molecular relapse as an increase of the MRD level of ≥1 log 10 between 2 positive samples in a patient who previously tested negative in technically adequate samples.

7. MFC-MRD Immunophenotyping is an essential, readily available tool for diagnosing AML and is currently the most commonly used MRD detection methodology. Two separate approaches have been used for assessing MFC MRD (2018): LAIP approach, which defines LAIPs at diagnosis and tracks these in subsequent samples The different-from-normal ( DfN ) approach, which is based on the identification of aberrant differentiation/maturation profiles at follow-up. The DfN approach can be applied if information from diagnosis is not available, and also to detect new aberrancies, together with disappearance of diagnosis aberrancies, referred to in earlier literature as “immunophenotype shifts. In 2021 guidelines, the panelist continues to recommend integration of diagnostic leukaemia-associated immunophenotype (LAIP) and different from normal ( DfN ) aberrant immunophenotype approaches.

8. (2021) :Some abnormal immunophenotypes may appear and/or disappear during monitoring, potentially because of transient expression on regenerating nonleukemic progenitors. This phenomenon may affect the respective specificities of both LAIP and DfN MRD detection, in particular when the percentages of LAIPs at lower thresholds ( eg , <0.1%) are investigated. Particular attention should be devoted to evaluating expression of the identified aberrant immunophenotypes in control samples that include regenerating BM (recommendation A4). When immunophenotypic abnormalities in specific samples could reflect transient features of regenerating or stressed hematopoiesis, the MRD report should comment on this possibility and note that a repeat sample in 2 to 4 weeks, if clinically indicated, may be informative.

9. Both approaches require expertise in the recognition of aberrant populations and exclusion of potential background as part of assay validation. Ideally, a diagnostic sample is preferred to determine whether a patient has diagnostic flow cytometric MRD targets that can be tracked. Markers for MRD assessment Implementation of a common, minimum required set of tubes/fluorochromes is a prerequisite for harmonized MRD detection, analysis, and reporting ( e.g 8 colors) Harmonized use of the integrated diagnostic-LAIP and DfN strategy for MRD detection that incorporates core MRD markers CD34, CD117, CD45, CD33, CD13, CD56, CD7, and HLA-DR to assess all samples. Some investigators favor addition of CD38 whenever possible, as CD38 adds specificity to certain aberrant leukemic immunophenotypes, particularly for the CD34 + CD38 low− compartment, when markers such as CD56, CD7, and others, such as CD45RA, designated as leukemic stem cell markers, are aberrantly expressed. In cases with a monocytic component, additional markers ( eg , CD64, CD11b, and CD4) may also be relevant.

10. Sampling and preanalytical phase: technical requirements (2021) Strong recommendations of first pull BM (EDTA or heparin tube, less than 5ml to prevent hemodilution from PB- presence of >90% mature neutrophils in a BM sample indicating significant hemodilution ) Should be processed undiluted within 3 days of storage at ambient For samples stored at ambient temperature >3 days, the MRD report should make specific note of sample quality and potentially compromised cell viability. Sample preparation can be performed using 2 accepted techniques: bulk lysis, followed by wash/stain/wash (all tubes prepare similar way for different stain) stain/lyse/wash or no wash (reduces cell loss compared to (1) ) technique selected should reliably produce high-quality MFC measurements ( ie , optimal cell concentration and no loss of forward scatter [FSC] or side scatter [SSC] properties) and should be applied consistently across samples.

11. Gating strategies and calculations of MRD (2021) Acquisition-a cquire >500 000 CD45 expressing cells and at least 100 viable cells in the blast compartment assessed for the best aberrancy( ies ) available. Use a gating syntax including FSC vs time and doublet exclusion plots ( eg , FSC-area vs FSC-height). Viability can be assessed by the addition of a viability dye or simply by accurate gating based on physical parameters (low FSC vs low SSC). To reliably use flow MRD for clinical decision making, studies of the lower limit of detection (LLOD) and lower limit of quantification (LLOQ) are essential. Thus, the panel recommends that LLOD and LLOQ should be calculated to assess MFC-MRD assay performance (recommendation A12) for each panel combination used. This statement aligns with the advice of regulatory agencies, which emphasizes that reporting MRD − results without LLOD information is not meaningful.

12. How to calculate MRD burden and minimal requirements (2018) Use LAIPs that clearly occupy an empty space, that is, aberrancies not found at the same MFC location in control BM, at diagnosis and follow-up. In cases where only part of a population is occupying an empty space, inclusion of additional cells outside the empty space is allowed provided they define 1 single clustered population together with the cells from the empty space. Use the best (most specific and/or highest frequency) LAIP for assessing MRD frequency; in case of multiple, nonoverlapping LAIPs, frequencies of individual LAIPs should be added up. Relate LAIP events to the leukocyte population of CD45 + cells (excluding CD45 − erythroblasts). Use the diagnosis LAIPs if diagnosis sample and diagnosis LAIPs are available to optimally inform MRD gating for these LAIPs. Use the DfN approach to identify any new LAIPs. Such new LAIPs can be used for quantitation.

13. MRD reporting (2018) (2018)- recommends using 0.1% as the threshold to distinguish MRD-positive from MRD-“negative” patients. However, it should be noted that MRD tests with MRD quantified below <0.1% may still be consistent with residual leukemia, and several studies have shown prognostic significance of MRD levels below 0.1%. Thus, cutoff levels below 0.1% ( eg , <0.01%) may define patients with particularly good outcome.

14. Elements in an MRD report should contain the following parameters (a) Absolute numbers of LAIP cells and WBCs, and LAIP cells as percentage of WBCs; (b) for diagnostic LAIPs, the percentage coverage of blast cells at diagnosis; and (c) clinicians and laboratory staff should collaborate to decide if the final report will contain a statement “MRD-positive” or “MRD-negative” ( ie , MRD ≥0.1% or < .1%). In cases with complete absence of aberrancies, the term “no MFC MRD identified” can be added to report of “MRD negative.” Detection sensitivity threshold for the aberrancy used with details: all aberrancies have the 0.1% threshold level, but additional information about the particular nature (sensitivity/specificity) of an aberrancy may be important, for example nature of myeloid, primitive, aberrant, and exclusion marker, especially in cases of newly defined LAIPs not present at diagnosis. Comments on quality of the sample, for example viability, insufficient regeneration, and PB contamination (Figure 1B). For suboptimal samples with detectable MRD, numbers of LAIP + cells need to be communicated. It is up to the clinician (or clinical study group) how to deal with information for MRD <0.1%: the report could contain “MRD detectable but <0.1%, may be consistent with residual leukemia” but also the statement “this level has not been clinically validated” when applicable for the laboratory involved. Alternatively, MRD <0.1% may be reported as “MFC MRD detectable and quantifiable, but with uncertain significance.” Leaving out such information may have medico-legal consequences.

15. Clinical Implementation (2021) MRD as a prognostic risk factor MRD should be assessed to refine relapse risk in patients who achieve morphologic remission, with full or partial hematologic recovery (CR/ CR i / CR p / CR h ) MRD thresholds MFC -0.1 %, qPCR - cycling threshold <40 in ≥2 of 3 replicates (recommendation D12). MRD test negativity by qPCR is defined as a cycling threshold ≥40 in ≥2 of 3 replicates, when at least 10 000 copies (but optimally, ≥30 000 copies) of the housekeeping gene ABL1 (or comparable numbers for other housekeeping genes, eg , GUS and B2M ) were measured

16. Optimal time for MRD