The value of measurable residual disease (MRD) assessment in acute myeloid leukaemia

Acute myeloid leukaemia (AML) is an aggressive haematopoietic stem cell malignancy, and the most common form of acute leukaemia in adults – with an incidence rate of 2–6 cases per 100,000 globally.¹

• Following treatment, ∼50% of patients who achieve complete remission (CR) will relapse, thereafter facing a poor prognosis²
• In relapsed AML cases, mutational screening/cytogenetic analysis can be highly beneficial to interrogate the evolving clonal landscape of the disease³
• Measurable residual disease (MRD) denotes the presence of leukaemia cells, including residual leukaemic clones, and can aid in establishing a myriad of patient-centred factors including remission status, relapse potential and refinement of therapeutic interventions

Next-generation sequencing (NGS) has emerged as a promising approach to detect MRD with high sensitivity, providing a highly reproducible, quantitative and easy to QC assay that can be deployed in clinical labs to inform patient prognoses, guide MRD status-adapted treatment strategies and to evaluate the efficacy of therapies.

Understanding MRD in AML

Published analyses have documented the prognostic relevance of MRD in acute myeloid leukaemia (AML), which has highlighted that those who were defined as MRD negative had a better 5-year survival rate compared to those defined as MRD positive.^4,5

However, current methods for the detection of MRD in AML can have a limited ability to provide a full picture of a sample’s MRD status because they focus on specific biomarkers. This may fail to capture and address the broader genomic heterogeneity present in AML, as leukaemic cells exhibit an extreme level of biological heterogeneity, displaying diverse genetic markers both within and across individuals, which can vary at different treatment stages.

Figure 1. Example clonal evolution pattern in a 74-year-old man with newly diagnosed therapy-related acute myelomonocytic leukaemia. Figure adapted from Morita K et al. Nat Commun 2020;11:5327⁶ (CC BY 4.0 (http://creativecommons.org/licenses/by/4.0))

Generally, CR patients who are MRD positive have an increased rate of relapse and a lower overall survival rate, compared to MRD negative patients given the same treatment.⁷ In addition, MRD assessment is also of value when evaluating patients for treatment via allogeneic HSCT. A positive MRD status was noted as a significant indicator (p<0.001) of AML relapse following HSCT, particularly if performed during a patient’s second period of disease remission.

As such, the detection of MRD is pivotal for establishing a deeper understanding of a patients AML remission status, especially at levels beyond the current morphology-based criteria. Indeed, organisations such as the European LeukemiaNet MRD Working Party supports MRD as one of the strongest independent assessments for AML prognosis, monitoring and treatment.⁸

Limitations of conventional MRD assessment methods

Traditionally, MRD assessment has been performed via multiparametric flow cytometry (MFC) or reverse transcriptase-quantitative PCR (RT-qPCR).

PCR-based detection

This method amplifies DNA so that specific segments can be analysed allowing for genetic abnormalities to be detected from a very small number of cancer cells with a high degree of sensitivity.

This method may be limited in MRD assessment by the fact that it can only be used for the detection of individual biomarkers per run. Given the extreme heterogeneity of leukaemic cells between individuals, and the instability of some markers during the course of disease and treatment, this approach can lead to false negative testing, since biomarkers that were present at the onset of disease may no longer be indicative of AML disease presence following treatment, or in relapsed patients.

Figure 2. PCR-based detection of MRD.

Multiparametric flow cytometry (MFC)

MFC interrogates immunophenotypically abnormal cell populations in a sample by labelling specific intracellular and cell surface markers. This can allow the determination of MRD through leukaemia-associated phenotypes or “difference-from-normal” phenotypes in white blood cells measured from samples.⁹

MFC is much more widely applied compared to PCR-based approaches, however in approximately 10% of cases, patients with AML do not exhibit abnormal immunophenotypes. Additionally, there is a lack of standardisation in monoclonal antibody panel composition and varying MFC protocols between institutions—which make the approach difficult to harmonise between laboratories.¹⁰

Figure 3. MFC-based detection of MRD. 

The benefits of NGS for MRD assessment

NGS-based MRD simultaneously queries, and can be used to analyse, the mutational status of a large number of disease-associated genetic variants associated with AML.

By testing for multiple variants in a single run, it is possible to rapidly gain a stronger understanding of a patient’s somatic mutation profile and the sequencing depth available through NGS enables MRD to be detected with improved sensitivity versus conventional methods, detecting variant allele fractions (VAFs) at ultra low levels.¹¹ Aided by this sensitivity, NGS-based methods can provide clearer stratification of individuals and help inform us about their prognostic and therapeutic response characteristics.

As NGS is today being more widely implemented for oncology testing, many clinical labs already possess much of the infrastructure required for MRD NGS assays. The multiplexing capabilities of NGS enable the capture of multi-gene information for multiple patients in a single run, reducing the analytical cost per individual.

A well-developed NGS assay can have broad applicability for patients with AML, and can be implemented in large-scale clinical studies. Overall, NGS currently stands as a comprehensive, convenient and sensitive means of detecting MRD in AML.

Introducing the SureSeq Myeloid MRD Panel

To fully realise the potential of NGS for MRD assessment, at OGT we have combined our decades of in-house experience in molecular biology and clinical haematology with insights from leading cancer KOLs, to target a comprehensive range of disease-associated genes, for MRD assessment.

The SureSeq™ Myeloid MRD Panel is the latest addition to OGT’s myeloid portfolio that delivers outstanding sensitivity, down to 0.05% VAF, and superior coverage uniformity, even for challenging targets such as NPM1 and CEBPA, as well as very large FLT3 internal tandem duplications (ITDs).

Encompassing 45 hotspot exons associated with genetic variants in AML, the guideline-oriented panel design allows for broad applicability of testing by simultaneously querying all disease-relevant variants in a single assay.

Our PCR-independent process improves coverage uniformity¹², and allows for the capture of low-abundance targets, while eliminating the risk of PCR bias and artifacts associated with amplicon sequencing. It is particularly advantageous for detecting low frequency variants, making the approach ideal for detecting MRD in widely heterogenous AML samples.

The SureSeq Myeloid MRD panel is easy to set-up and run in most research and laboratory settings. At OGT we help guide you through every step of your workflow and our dedicated support network includes our Field Application Scientists, who can visit your laboratory to give hands-on advice, and help to set-up and troubleshoot your NGS MRD assays.

Ready to transform your MRD assessments?

Our SureSeq Myeloid MRD Panel delivers a comprehensive panel with superior design as standard. At OGT, we provide the tools, expert support and streamlined workflow to help you to get your MRD research up and running.

Connect with us today to discuss your MRD project needs.

References

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4. Moritz J et al. Biomedicines 2024;12:599. doi: 10.3390/biomedicines12030599
5. Short NJ et al. JAMA Oncol 2020;6:1890–1899. doi: 10.1001/jamaoncol.2020.4600
6. Morita K et al. Nat Commun. 2020;11:5327. doi: 10.1038/s41467-020-19119-8
7. Meddi E et al. Int J Mol Sci. 2023;24:3062. doi: 10.3390/ijms24043062
8. Schuurhuis GJ et al. Blood. 2018;131:1275–1291. doi: 10.1182/blood-2017-09-801498
9. Al-Mawali A et al. Am J Clin Pathol. 2009;131:16–26. doi: 10.1309/AJCP5TSD3DZXFLCX
10. Blachly JS et. Haematologica. 2022;107:2810–2822. doi: 10.3324/haematol.2022.282034
11. Vonk CM et al. Cancers. 2021;13:5431. doi: 10.3390/cancers13215431
12. Akabari R et al. Genes. 2022;13:630. doi: 10.3390/genes13040630

SureSeq: For Research Use Only; Not for Diagnostic Procedures.