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Measurable Residual Disease (MRD) in Acute Myeloid Leukemia (AML)

AML Overview

Acute myeloid leukemia (AML) is a highly heterogenous group of malignancies, driven by various mutations and/or chromosomal aberrations. Affecting the blood and bone marrow, the disease is characterized by the clonal expansion of immature "blast cells" that leads to ineffective hematopoiesis (blood cell formation) and ultimately bone marrow failure.1

While most AML patients achieve remission following induction chemotherapy, disease relapse is common2. The 5-year survival rate for people over 20 years of age is only 27%.3 Each progressive relapse may lower the probability of long-term survival2.

 

AML Relapse

AML relapse generally arises from the residual presence of a small number of the original leukemic clones or the emergence of a new clone that is genetically similar. In the latter case, a leukemic clone can acquire resistance mutations and develop survival advantages that can drive disease relapse4. Hence, it is important to eradicate even the smallest number of leukemic clones in order to achieve a cure.

Identification of these remaining clones is termed measurable residual disease (MRD) detection.2 Studies have shown that MRD may be a strong predictor of relapse and survival, and can have future implications on therapeutic strategies in the management of AML5,6. MRD is also commonly employed as a surrogate end point in clinical trials to help accelerate development of novel regimens.2

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Traditional MRD Detection in AML Samples

Assessment of MRD in AML samples is challenging due to the need for highly sensitive detection of small amounts of residual disease, coupled with the need to cover a variety of biomarkers, given the high degree of disease heterogeneity. Several technologies are available, but traditional approaches have key limitations and lack standardization and comparability7.

Flow Cytometry

MRD detection by multi-parameter flow cytometry (MFC) relies on the analysis of antigens associated with leukemic cells that are infrequent in healthy bone marrow. This method does not track individual mutations like qPCR or NGS. The advantages of MFC is that it is widely available in many laboratories and offers relatively rapid turnaround times8.

The major drawback is that the interpretation of the results is very subjective, making the technique difficult to standardize across different laboratories. A high degree of expertise and experience is required for MRD analysis, which is not universally available in all laboratory settings.

qPCR and dPCR

Unlike MFC, qPCR based MRD detection can track genetic alterations present in leukemic clones, including chimeric fusions, gene rearrangements, genetic alterations, and differentially expressed genes. Using this approach, the data can be more accurately quantified, which obviates the challenge of subjective interpretation that hampers MFC. This technique can be widely deployed in a variety of molecular testing laboratories.

More recently, digital PCR (dPCR) has emerged as a potential MRD detection method. The fundamental difference is that it partitions the PCR reactions into thousands to millions of small droplets, whereby each droplet can contain a single copy of the target template. This enables a digital output based on counting of individual droplets, which can accurately quantify of the frequency of the mutant alleles relative to the wildtype alleles.

The primary drawback of PCR is that it only enables labs to track a very limited number of targets, so a specific set of primers must be selected for each individual sample. Furthermore, for many less-common mutations, an MRD PCR test is often not commercially available. Given the high degree of genetic heterogeneity in AML, it would be advantageous to have a universal assay that can track a of broad range of mutations to account for both known targets and potential new ones that can emerge through the course of disease.

 

NGS: An Emerging Technique for MRD Analysis

Significant research efforts are currently ongoing to develop better standards and evaluate new genetic testing methods for MRD in AML. Next-generation sequencing (NGS) technology is an emerging technique that holds great promise for MRD assessment. Like PCR, NGS can track a range of genetic alterations and the analysis is not reliant on subjective interpretation like MFC. The fundamental advantage is that NGS can evaluate many genetic targets simultaneously, so that labs can assess a wide range of mutations across multiple different samples with a single universal test.

Assessing a wide range of mutations in important because clonal evolution in AML can occur through the acquisition of additional mutations that were not present in the original leukemic clone, which can become drivers for disease relapse4. Techniques that only track known mutations can miss acquired mutations in emerging sub-clones. For example, a recent study demonstrated that some NPM1 mutations observed at diagnosis were not present in relapsed clone.9

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Traditionally, NGS was limited by relatively low sensitivity due in part to standard error rates in sequencing data. In addition, the technology and bioinformatic analysis required specialized expertise only available in a limited number of centers.

However, new techniques that leverage molecular barcoding strategies enable NGS tests to now achieve a high level of sensitivity required for MRD detection. Advances in workflow automation have made NGS testing much easier to implement in standard laboratories without requiring specialized expertise.

 

The Oncomine Myeloid MRD Assay (RUO)

 

 

To help advance important MRD research in AML and other myeloid neoplasms, Thermo Fisher Scientific has introduced the Oncomine Myeloid MRD Assay (RUO). This advanced NGS test offers an MRD-specific DNA and RNA panel, which enables labs to track DNA-level genetic alterations and chimeric gene fusions simultaneously.

The panel content covers genes associated with a range of myeloid neoplasms, including AML, myelodysplastic syndromes (MDS), and myeloid proliferative neoplasms (MPN). Using highly sensitive AmpliSeqHD technology, the assay can achieve a low limit of detection, down to 0.05% allele frequency.

The test includes a full analytically validated informatics pipeline and reporting tools. This tool set includes a specialized workflow for detecting challenging FLT3-ITD mutations commonly present in AML and offers capabilities for longitudinal tracking of target variants.

Learn more about the Oncomine Myeloid MRD Assay (RUO)

For Research Use Only. Not for use in diagnostic procedures.

References
1. Akiti A et al. Acute Myeloid Leukemia. [Updated 2021 Aug 17]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK507875/
2. Aitken MJL et al. J Hematol Oncol. 2021; 14:137
3. https://www.cancer.net/cancer-types/leukemia-acute-myeloid-aml/statistics
4. Vosberg S et al. Genes Chromosomes Cancer. 2019; 58:839–849
5. Kantarjian H. Am J Hematol. 2016; 91(1):131–45.
6. Heuser M et al. Blood. 2021; 38(26): 2753-2766
7. Ngai LL et al. 2021; Front. Oncol. 10:603636
8. Voso MT et al. Frontiers in Oncology. 2019; 9:665
9. Hollein A et al. Blood Advances. 2018; 2(22): 3118-3125

 

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