Genomic Alterations in Acute Myeloid Leukemia in Routine Clinical Practice

Review Article

Ann Hematol Oncol. 2022; 9(4): 1405.

Genomic Alterations in Acute Myeloid Leukemia in Routine Clinical Practice

Benková K1,2, Jelínek T1,2, Hájek R1,2 and Korístek Z1,2*

1Department of Hematooncology, University Hospital Ostrava, Ostrava, Czech Republic

2Department of Hematooncology, Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic

*Corresponding author: Zdenek Korístek, Department of Hematooncology, Faculty of Medicine, University Hospital Ostrava, 17. listopadu 1790/5, Ostrava 708 52, Czech Republic

Received: August 17, 2022; Accepted: September 16, 2022; Published: September 23, 2022


Acute Myeloid Leukemia (AML), the most common acute leukemia in adults, is a genetically heterogeneous disease. Genomic alterations condition the pathophysiology of AML. Nowadays, these changes are considered to be important biomarkers for risk stratification, treatment decisions (including new targeted drugs) and Minimal Residual Disease (MRD) monitoring during followup of AML. Positive MRD in AML patients is associated with a higher risk of relapse and shorter overall survival compared to MRD negative individuals. Nevertheless, MRD-targets, diagnostic techniques, time points of MRD determination, and analyzed material are not standardized. Thus, integration of MRD testing in individual AML patients in routine practice is still a workin- progress. This review article comprehensively focuses on key molecular biomarkers in AML and their utilization in clinical practice, especially regarding risk assessment and MRD testing. Moreover, new AML molecular-targeted therapies are briefly summarized here.

Keywords: Acute myeloid leukemia; Genomic alterations; Molecular biomarkers; Minimal residual disease; Targeted therapies


Acute myeloid leukemia is characterized by uncontrolled proliferation and accumulation of clonal immature myeloid cells in bone marrow and peripheral blood leading to hematopoietic failure [4]. AML is a genetically heterogeneous disease with a very variable prognosis and high mortality rate. The 5-years Overall Survival (OS) is less than 50% and only 20% of elderly patients will survive 2 years after diagnosis [48]. Nowadays, cytogenetic profile and new molecular markers play an important role in diagnosis, prognosis, and monitoring of AML, as well as in optimizing therapeutic strategies [48].

Achieving complete remission defined by the absence of leukemic blasts in the bone marrow is not sufficient for predicting longterm remission as most patients relapse [63]. This prediction was improved with measuring the residual levels of leukemic cells (also called minimal or measurable residual disease; MRD) that persist in the bone marrow after chemotherapy [41]. In current studies, MRDtargets, diagnostic techniques, time points of MRD assessment, and analyzed material are variable. With the exception of MRD detection in Acute Promyelocytic Leukemia (APL), for which standardized guidelines have been published [54], there is still a lack of prospective randomized studies providing data on how to modulate treatment to alter outcomes based on MRD status in non-APL AML. It has been proven that MRD assessment allows outcome prediction. So far, implication of MRD testing in individual AML patients in routine practice is still a work-in-progress [28].

In this work, we comprehensively review key molecular biomarkers in AML, focusing on their utilization in clinical practice. We emphasize risk stratification and MRD testing, as well as novel AML targeted therapy. Routinely used methods to determine MRD and some future perspectives, and finally, practical aspects of MRD monitoring are also briefly presented.

Techniques for MRD Assessment

In practice, MRD evaluationis commonly performed by two methods – Multicolor Flow Cytometry (MFC) and real time quantitative PCR (RT-qPCR). Modern techniques such as Next Generation Sequencing (NGS) and digital droplet PCR (ddPCR) could be applied to routine clinical practice in the near future on account of their benefits [28].

Multicolor Flow Cytometry

Multicolor flow cytometry is a technique recognizing antigens on leukemic cells by fluorescence-labeled antibodies [28]. MRD detection by MFC is rapid and applicable to almost all AML patients (up to 90%), but has lower sensitivity (10-3–10-4) when compared to other techniques [57,67]. Two partially overlapping analyzing strategies can be used for MFC-MRD; the first focuses on Leukemia- Associated Immunophenotypes (LAIP), which are determined at the time of diagnosis and then used to track down residual leukemic cells in the follow-up samples. The second one is based on identifying any immunophenotypes that are Different-from-Normal (DfN) in samples submitted for MRD analysis [18,28,57,67]. Currently, the integrated LAIP-based DfN approach is recommended by the European Leukemia Net (ELN) MRD Working Party [56]. Unfortunately, MRD monitoring by MFC has not been fully standardized in AML in comparison to other hematologic malignancies [27].

PCR-Based Methods

Polymerase Chain Reaction (PCR) is a method that allows quantification of nucleic acids by amplification with the enzyme DNA polymerase [8]. In AML, these techniques identify AML-associated mutations in the follow-up samples. Real-time quantitative PCR is the golden standard for molecular MRD detection. RT-qPCR provides high sensitivity (10-4–10-6) and specificity, however, it can be used only in those patients harbouring specific aberrations (especially NPM1, CBF-MYH11, RUNX1-RUNX1T1, PML-RARA, accounting for approximately 40% of all AML patients) [18].

An innovative method called droplet digital PCR provides high precision quantification which is very reliable and offers the possibility to monitor several mutations simultaneously. However, it is not currently being used in routine practice, mainly due to higher costs and a limited number of equipped laboratories [8,18,28].

Next Generation Sequencing

NGS allows us to increase our knowledge of molecular heterogeneity in AML. Due to broader mutation coverage, it has the potential to be used in almost all AML patients. However, the sensitivity of NGS assays depends on DNA quality and quantity, as well as on the distinct monitored mutations. Universally standard quality criteria for NGS have still not been determined. Therefore, its application in routine practice has to be individually validated [1,28,57,66].

Molecular Biomarkers

FMS-Like Tyrosine Kinase 3 (FLT3)

FMS-like tyrosine kinase 3 is a transmembrane ligand-activated receptor tyrosine, expressed by the hematopoietic stem or progenitor cells, which regulates the differentiation, proliferation, and survival of these cells. Mutations in the FLT3 gene cause dysregulation of the gentle balance between cell proliferation and differentiation.

FLT3 mutations are found in approximately 30% of newly diagnosed AML cases. FLT3 internal tandem duplication (FLT3- ITD), the most frequent mutation (approximately 25% of AML cases) significantly affects the prognosis of AML. Point mutations in the tyrosine kinase domain (FLT3-TKD) have a lower incidence in AML (approximately 7 – 10% of all cases) and their prognostic value is uncertain [9,21,48,]. FLT3-ITD mutations are associated with high White Blood Ccell (WBC) counts, high percentage of peripheral and bone marrow blasts and represent a negative risk factor for overall survival and Event-Free Survival (EFS) [21,47].

FLT3 detection methods include agarose gel electrophoresis following PCR-electrophoresis and fragment analysis using capillary sequencing [53]. Approximately 25% of FLT3-ITD negative relapses are recorded in patients with FLT3-ITD positivity at the time of diagnosis. FLT3-ITD mutation is unstable and nowadays it is not consider being a reliable MRD marker [35].

The prognosis of AML patients with FLT3-ITD mutations is significantly affected by several other factors: the Allelic Ratio (AR) of FLT3-ITD to wildtype FLT3, karyotype, and the presence of nucleophosmin 1 mutations (NPM1) [9,49].

A higher FLT3-ITD allelic ratio (AR>0.5, FLT3-ITDhigh) is associated with higher relapse rates, refractory disease, and shorter OS [16]. Allogeneic hematopoietic stem cell transplant (allo-HSCT) in the first Complete Remission (CR) shows improved Relapse- Free Survival (RFS) and OS in patients with AR>0.5compared to patients receiving standard intensive chemotherapy with or without autologous HSCT. This effect was not seen in patients with low allelic ratio (AR<0.5, FLT3-ITDlow) [55].

Both FLT3-ITD and FLT3-TKD mutations are associated especially with normal cytogenetics (CN-AML) or t(15;17), and rarely with complex karyotype or CBF-leukemia (CBF-MYH11 and RUNX1-RUNX1T1) [42,47]. Patients with t(15;17) acute promyelocytic leukemia harbouring FLT3 mutations are more likely to present with elevated WBC counts and poorer prognosis than those without mutations [45].

The most recent studies propose that patients with FLT3-ITDlow and NPM1 mutations have similarly favorable outcomes as patients with NPM1 mutations without FLT3-ITD.Conversely, patients with FLT3-ITDhigh and NPM1 wild type have a poor prognosis [14]. These are key changes in revisited risk stratification of AML 2017 (Figure 1) [14]. Allogeneic HSCT in CR1 should benefit for patients with intermediate and high risk AML according to the time-dependent analysis [5].

Citation: Benková K, Jelínek T, Hájek R and Korístek Z. Genomic Alterations in Acute Myeloid Leukemia in Routine Clinical Practice. Ann Hematol Oncol. 2022; 9(4): 1405.