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Clinical Utility of Individualized Follow-Up in Acute Myeloid Leukemia (AML) Patients Using a Myeloid NGS Panel Volume 53- Issue 3

María José Larráyoz1,2#, Almudena Aguilera Díaz2,3#, Zuriñe Blasco Iturri1, Iria Vázquez1,2, Beñat Ariceta2,3, Amagoia Mañú1,2, Paula Aguirre Ruiz2,3, Maria Cruz Viguria2,4, María Teresa Zudaire2,4, Eva Bandrés2,4, María del Carmen Mateos2,4, José Rifón Roca2,5, Ana Alfonso Piérola2,5, Felipe Prósper2,3,5, Marta Fernández Mercado1,3*# and María José Calasanz1,2,6#

  • 1Hematological Diseases Laboratory, CIMA LAB Diagnostics, University of Navarra, Spain
  • 2Navarra Institute for Health Research (IdiSNA), Spain
  • 3Advanced Genomics Laboratory, Hemato-Onclology, Center for Applied medical research (CIMA), University of Navarra, Spain
  • 4Hematology Service, Hospital Universitario de Navarra (HUN), Spain
  • 5Hematology Department, Clinica Universidad de Navarra (CUN), Spain
  • 6Scientific co-Director of CIMA LAB Diagnostics, CIMA LAB Diagnostics, University of Navarra, Spain
  • #These authors contributed equally to this work

Received: September 07, 2023; Published: October 25, 2023

*Corresponding author: Marta Fernández Mercado, Advanced Genomics Laboratory, Hemato-Oncology, Center for Applied medical research (CIMA), University of Navarra, Pamplona, Spain

DOI: 10.26717/BJSTR.2023.53.008401

Abstract PDF


The current standard to determine complete remission in acute myeloid leukemia (AML) includes morphologic criteria, cytogenetics, classical molecular analysis -such as polymerase chain reaction (PCR) and quantitative reverse-transcription PCR (RT-qPCR), and flow cytometry. Recently, Next- Generation Sequencing (NGS) is being a useful tool to gain insights into the molecular alterations in patients diagnosed with AML. We hypothesized that deeper knowledge of the clonal dynamics of AML could potentially be of clinical utility. We studied with our custom Pan-Myeloid NGS Panel the genomic alterations of 71 samples, corresponding to 20 AML cases during disease follow-up. Sequencing data identified genomic clonal markers with clinical utility in 90% of cases.

In patients not receiving hematopoietic stem cell transplant (HSCT) (n=6), NGS classified patients in two genetic profiles:

(i) Those with negative measurable residual disease (MRD).

(ii) Those not responding to treatment and undergoing disease progression (these, with pathogenic variants in DDX41, DNMT3A, IDH1, JAK2, NRAS, SRSF2, U2AF1 genes).

In patients receiving HSCT (n=14), NGS was useful to classify them into three groups:

(i) Patients not presenting clinically relevant variants.

(ii) Patients clearing pathogenic variants upon HSCT.

(iii) Patients with persistent variants after HSCT. Interestingly, NGS data detected clones harboring pathogenic variants in two patients with negative MRD by flow cytometry, indicating that NGS could complement the current gold standard follow-up method in some instances.

Keywords: AML; NGS; MRD; Variants; Follow-Up

Abbreviations: AML: Acute Myeloid Leukemia; PCR: Polymerase Chain Reaction; NGS: Next-Generation Sequencing; HSCT: Hematopoietic Stem Cell Transplant; MRD: Measurable Residual Disease; CR: Complete Remission; WES: Whole Genome Sequencing; PMP: Pan-Myeloid Panel; UN: University of Navarra; ACMG: American College of Medical Genetics and Genomics; IGV: Integrative Genomics Viewer; VUS: Variant of Uncertain Significance


Acute Myeloid Leukemia (AML) is a genetically heterogeneous neoplasm characterized by the accumulation of blasts due to genetic alterations in hematopoietic stem and/or progenitor cells [1,2]. These malignancies are one of the most common in adults and tend to be more aggressive than other leukemias [3]. Approximately 70% of AML patients achieve morphologic complete remission (CR) after chemotherapy; unfortunately, 50% of these patients eventually relapse [4-6]. Therefore, the course of these patients needs to be followed up in deeper detail, in order to tailor the therapeutic strategies to the specific clonal evolution of each case. The diagnosis of AML is a multidisciplinary process that integrates the results of different techniques including cytomorphology, flow cytometry, cytogenetics, and molecular biology. Specifically, cytogenetic and molecular studies are essential to determine risk groups, guide treatment, and define markers of follow-up to detect measurable residual disease (MRD) [7-9].

The current availability of immunophenotyping and/or molecular markers allows determining the kinetics of the disappearance of the disease, design of individualized post- remission treatment strategies, and early detection of relapse. Molecular detection MRD is classically carried out by reverse transcription quantitative polymerase chain reaction (RT-qPCR), due to the high sensitivity of this technique (10-4-10-6) [10]. In the last few years, Next Generation Sequencing (NGS) is being increasingly used for the genomic characterization of clinical samples. Whole Genome Sequencing (WES) and Whole Genome Sequencing (WGS) studies have shown that 70% of AML patients present somatic mutations [1,2,11]; moreover, mounting evidence shows that clinically relevant mutations are associated with an increased risk of relapse and reduced overall survival [6,9,12]. For example, internal tandem duplications in FLT3 gene (FLT3-ITD), partial tandem duplications in KMT2A (MLL) gene (MLL-PTD), and mutations in ASXL1, RUNX1, and TP53 genes are associated with shortened overall survival [7,13], to name a few. The number of clinically relevant genes in AML is already more than 30, and these numbers, as well as our understanding of this pathology, are growing mostly thanks to NGS technologies [14].

In particular, NGS gene panels, due to their focus on specific genes related to the pathology in study, have been shown to achieve a high sequencing depth (1000-5000x), which greatly improves sensitivity over traditional Sanger Sequencing [15] and is more sensitive than other NGS techniques such as WES [16]. This, together with the fact that NGS costing is lower and the turnaround time is shorter than individual Sanger sequencing testing, makes NGS panels ideal tools for molecular monitoring of AML patients [2,17-19]. The present study aims to evaluate the performance of a custom Pan-Myeloid Panel (PMP) (48 genes, SOPHiA GENETICS), in the monitoring of 20 patients diagnosed as or progressing to AML during the course of the disease, the majority of them receiving hematopoietic stem cell transplant (HSCT, n=14). The characterization of the dynamics of the molecular architecture of these patients over time was then correlated to treatment efficacy in order to evaluate the clinical utility of this technique.

Materials and Methods

Sample Collection

We collected 71 bone marrow samples at different stages of the disease (diagnosis, post-treatment, post-HSCT) from 20 patients diagnosed with AML (13 de novo, and 7 secondaries to a preexisting myeloid neoplasm) during disease follow-up; 14 of these patients received HSCT during the study (Table 1). The majority (n=17) of the first samples used in this cohort of patients were collected at the time of AML diagnosis. The remaining three samples were collected after relapse (Unique Patient Number 1, UPN1, and UPN13) and post-treatment (UPN9). All patients signed a written informed consent form for genetic testing, research, and tissue banking provided by the Biobank of the University of Navarra (UN) and were processed following standard operating procedures approved by the CEI (Comité de Ética de la Investigación) of UN. Patient data were fully anonymized, and all patients provided informed written consent to have data from their medical records such as age, gender, and diagnosis to be used for research purposes.

Table 1: Patient Cohort Description.


Note: UPN=Unique Patient Number; AML= Acute Myeloid Leukemia; Dx=Diagnosis; HSCT= Hematopoietic Stem Cell Transplant; ND=not done.

Sample Preparation

Genomic DNA from each sample was extracted using QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), quantified using Qubit dsDNA BR Assay Kit on a Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and DNA quality was assessed by DNA genomic kit on a Tape Station 4100 (Agilent Technologies, Santa Clara, CA, USA).

Pan-Myeloid Panel (PMP), Alignment, and Variant Calling

Our custom Pan-Myeloid Panel (PMP) is a hybridization capture-based panel that counts on a total genomic footprint of 114 kb, targeting 63 genes. For the detection of Single Nucleotide Variants (SNV), insertions and deletions (indels) we targeted 48 genes: full CDS of 22 genes, and exonic hotspots of 26 additional genes [20]. NGS libraries were prepared following manufacturer’s instructions (SOPHiA GENETICS, Saint Sulpice, Switzerland). Final NGS libraries were quantified using Qubit dsDNA HS Assay Kit in a Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and quality was assessed using DNA D1000 kit and visualized on Agilent 4100 Tape Station (Agilent Technologies, Santa Clara, CA, USA). A total of 10.5 pM of 8 pooled libraries was pair-end sequenced on a MiSeq (Illumina, San Diego, CA, USA) with 251x2 cycles using the Reagent Kit V3 600 cycles cartridge, according to the manufacturer’s instructions. FastQ files were directly obtained from the MiSeq and uploaded onto SOPHiA GENETICS DDM software (SOPHiA GENETICS, Saint Sulpice, Switzerland), where alignment, variant calling of SNV/indels, and annotation were performed.

Variant Data Analysis

The list of annotated variants was filtered to exclude intronic, intergenic, and synonymous ones. Two geneticists with expertise in hematological malignancies categorized variants according to current guidelines from the Spanish Group of Myelodysplastic Syndromes [21] and from the American College of Medical Genetics and Genomics (ACMG) [22]. Aligned reads were manually curated for confirmation of the presence of the filtered-in variants within the Integrative Genomics Viewer (IGV) software (Broad Institute) [23].


Overall, the results showed that 18 out of 20 patients presented at least one clinically relevant mutation (meaning pathogenic and likely pathogenic variants) throughout the time course of the disease. The most recurrent mutated genes were DNMT3A (35% of cases, 7 patients), FLT3 (30% of cases, 6 patients) ASXL1 (20%, of cases, 4 patients), SRSF2 (20% of cases, 4 patients), and NPM1 (20% of cases, 5 patients). We indeed found that in 4 cases mutations in NPM1 gene were concomitant with mutations in FLT3 gene (except UPN3), as it has extensively been described in the literature in AML [24]. NRAS and CBL genes were mutated in 15% of the patients. IDH2, CUX1, JAK2, RUNX1, WT1 and ZRSR2 genes were mutated in 2 cases each (10%); and finally, BCORL1, IDH1, DDX41, TET2, TP53, U2AF1, ETNK1, and PTPN11 genes were mutated in 1 case each (5%).

Variants Detected in Patients that did not Undergo HSCT

A total of 6 patients out of the 20 cases included in our study did not get HSCT, and all of them presented at least 1 pathogenic mutation (Table 2).

Table 2: Mutational Profile of patients that did not have HSCT.


Note: UPN=Unique Patient Number; Chr=Chromosome; Class=Classification; VAF=Variant Allele Frequency; Dx=Diagnosis; Tx=Treatment; HSCT= Hematopoietic Stem Cell Transplant; Lik. Path.= Likely Pathogenic; VUS=Variant of Uncertain Significance.

Patients Presenting Clinically Relevant Variant Clearing After Treatment: Amongst patients not receiving HSCT, 3 of them cleared all clinically relevant variants after treatment (UPN8, UPN11, and UPN12) (Figure 1). Of note, UPN8 did show persistent clonality after treatment as revealed by the presence of a Likely Pathogenic variant.

Figure 1


Patients With Positive MRD After Treatment: Out of the 6 patients that had been treated with the standard chemotherapy scheme, 3 of them presented positive MRD (Figure 2). Moreover, pathogenic mutations in NPM1 (UPN3) and KRAS (UPN20) genes appeared for the first time after treatment. In addition, we found that UPN19 presented two pathogenic mutations in DDX41 gene, having been this mutational pattern described as an indicative of a germline predisposition [25,26].

Figure 2


Variants Detected in Patients Who Underwent HSCT

A total of 14 out of the 20 patients included in our study underwent HSCT, and 11 of them (79%) presented at least 1 pathogenic mutation (Table 3). The first sample analyzed on the majority of the cases was collected at the time of diagnosis, except in three cases: UPN1 and UPN13 were firstly analyzed at relapse, and UPN9 was analyzed for the first time after treatment.

Table 3: Mutational profile of the patients that underwent HSCT.


Note: UPN=Unique Patient Number; Chr= Chromosome; Class= Classification; VAF= Variant Allele Frequency; Tx=Treatment; HSCT= Hematopoietic Stem Cell Transplant; VUS= Variant of Uncertain Significance; Lik. Path.= Likely Pathogenic.

Patients No Presenting Clinically Relevant Variants: The two patients who did not show any pathogenic variant were UPN1 and UPN2. UPN1 was a 6-year-old girl diagnosed with AML, who showed two variant of uncertain significance (VUS), one in RAD21 gene present since the time of relapse, and one in IKZF1 gene found only after HSCT relapse. UPN2 was a 23-year-old man, who only presented a VUS in BCORL1 gene at diagnosis, which was found to be cleared after HSCT (Figure 3). Although at this moment there is no clinical significance associated with these variants, we included them because it is possible that the continuous updating of the relevant databases might confer them a clinical meaning in the near future. Moreover, the presence of these variants is indicative of clonality, which might be useful for disease monitoring.

Figure 3


Note: UPN= Unique Patient Number; VUS= Variant of Uncertain Significance; HSCT= Hematopoietic Stem Cell Transplant.

Patients Presenting Clinically Relevant Variant Clearing After Treatment: Five patients presented clearance of all variants after treatment (UPN5, UPN6, UPN7, UPN9 and UPN10). Four of them were perfect examples of successful HSCT, showing complete disappearance of all variants, both pathogenic and VUS, upon HSCT (Figure 4). UPN6 was found with negative MRD after treatment, but still needed to be transplanted because of his complex karyotype (Table 1).

Patients with Positive MRD: A total of 7 patients showed a MRD upon HSCT, (UPN4, UPN13, UPN14, UPN15, UPN16, UPN17 and UPN18) (Figures 5 & 6). From these, in 5 cases the unsuccessful transplant had already been confirmed by positive MRD, as measured by flow cytometry. Interestingly, the remaining 2 cases (UPN4, UPN14) were found to present a negative MRD by flow cytometry, while NGS showed evidence of the presence of malignant clones (Figure 6). Remarkably, 3 of those 7 cases not only presented pathogenic mutations from the time of diagnosis that did not clear after treatment, but they also developed additional pathogenic variants for the first time after HSCT (UPN4, UPN13, UPN14).

Figure 4


Note: UPN= Unique Patient Number; HSCT= Hematopoietic Stem Cell Transplant.

Figure 5


Note: UPN= Unique Patient Number; HSCT= Hematopoietic Stem Cell Transplant.

Figure 6


Note: UPN= Unique Patient Number; HSCT= Hematopoietic Stem Cell Transplant; MRD= Measurable Residual Disease.


Our sequencing data on 71 samples from 20 AML patients diagnosed as or progressing to AML identified genomic clonal markers with clinical utility in 90% of cases. Eighteen out of 20 patients presented at least one clinically relevant mutation (average 2.6 mutations per patient); these, together with the mutation frequency observed in the present study, are in agree to previously published data in AML [14,27,28]. Identification of the pathogenic clones in these AML patients is crucial, due to the fact that a number of the detected mutated genes possess diagnostic, prognostic, and predictive value. For example, there is available targeted therapy for FLT3- ITD, IDH1, and IDH2 gene mutations, like Midostaurin or Gilteritinib, and Ivosidenib, and Enasidenib, respectively [9,29-31], that improve results associated with conventional therapy. Also, while mutations in NRAS genes have been related to progression of the disease [32], ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, TP53, U2AF1 and ZRSR2 have an adverse prognostic value [7,14].

Therefore, the genomic characterization of AML patients is indeed useful for therapeutic decisions and clinical management of patients, such as new cycles of consolidation chemotherapy, or different therapeutic interventions after HCST. Importantly, in this study NGS data in these 18 cases showed additional value when examined during the time course of the disease. We observed two different clonal genetic dynamic patterns: some patients presented persistent clinically relevant mutations after treatment (n=10), whereas some others showed pathogenic variants clearance after treatment (n=8). On the one hand, we have observed that 5 out of the 10 patients with persistent mutations, were found with new clones harboring additional pathogenic mutations after treatment on top of the founder pathogenic clone present from the time of the first diagnosis. On the other hand, in the remaining 5 cases all detected pathogenic clones were present from the time of diagnosis. In the first group, the mutations that sprang upon treatment failure affected NRAS, DNMT3A and KRAS genes.

In the second group, in 4 of the cases all the founding clones reduced their size after treatment, although the mutations were not totally cleared even after HSCT, which might be a sign of incipient early relapse, like indeed happened with UPN16 (Figure 5 & Table 3). The persistent pathogenic variants in these cases were located in BCORL1, DNMT3A, SRSF2, TET2 and DNMT3A genes in UPN15; ASXL1 and ZRSF2 genes in UPN17, and IDH1 in UPN18 (Table 3). For UPN19 there were variants that persisted during the time course of the disease with similar frequencies of those detected at the time of diagnosis. DDX41 gene presented 2 variants; DDX41 p.Gln604Hisfs*38 showed a stable VAF of ~50% in all samples, while DDX41 p.Arg525His presented a lower VAF that varied across the analyzed samples (Table 2). We suspected that the first variant could be germline since the VAF dynamic fits the double hit mutation pattern described by several studies in the DDX41 gene [25,26,33,34].

Indeed, specifically DDX41 p.Gln604Hisfs*38 variant has already been described to be of germline nature [26], although for this particular case it would be required to sequence a non-myeloid tissue (e.g. skin fibroblasts, hair follicles, or CD3+ cells) in order to check the presence/absence of the variant, since unraveling if the disease is originated on the grounds of a genetic predisposition is a crucial piece of information for making therapeutic decisions. Of note, we failed to detect any clinically relevant variants throughout the course of the disease in 2 cases (UPN1, UPN2) although variants of uncertain significance were present in both cases (Figure 3). UPN1 was a 6- year-old girl diagnosed with AML, who showed two VUS in RAD21 and IKZF1 genes at relapse. UPN2 was a 23- year-old man, who only presented a VUS in BCORL1 gene at the time of diagnosis, which was found cleared at follow up.

Therefore, even though the NGS panel failed to detect clinically relevant variants in these two cases, it was useful to show evidence of clonality, especially useful because these cases had no cytogenetic markers (i.e. were found to have a normal karyotype) (Table 1). For cases of this sort, NGS platforms with wider scope (e.g. whole exome sequencing, WES) might provide clinically relevant information, although with a more limited ability to detect minor clones, due to its reduced depth of coverage [16]. Remarkably, our NGS data detected clones harboring pathogenic variants in two patients with negative MRD as measured by flow cytometry. One of them was UPN4, a 58-year-old man diagnosed with AML who received HSCT. At diagnosis, the NGS panel detected a pathogenic variant in TP53 gene (VAF 76%) and after induction treatment, this clone drastically reduced in size (VAF 1%); at day +28 post HSCT, the pathogenic clone harboring the TP53 gene mutation could not be detected (VAF 0%; 7641x depth), but at day +180 post HSCT, it was found to have expanded (VAF 2%); at day +306 post HSCT it was detected the TP53 gene mutation (VAF 65%) and concomitantly a pathogenic variant in NRAS gene appeared (VAF 6%).

At post-induction, when the NGS panel detected the pathogenic clone, flow cytometry tested negative for MRD, in clear disagreement with molecular data. The other case was UPN14, a 72-year-old female patient with a VUS in DNMT3A gene that, even though it has not been shown to be clinically relevant, still was useful to reveal clonality, proving that the transplant had not been successful, although flow cytometry failed to measure any residual disease. Moreover, at day +186 post HSCT we could detect an additional clone with a different mutation, also in DNMT3A gene (Figure 6 & Table 3); remarkably, flow cytometry tested negative for MRD throughout the disease course. These cases are two examples that illustrate how NGS can be a good complement to standard MRD techniques which are already in place in the clinical setting, as has been suggested before [4,9,35].

Overall, our myeloid NGS panel was an excellent tool for the genomic characterization of AML patients during the time course of the disease, since it identified variants that are related to the pathogenicity of the disease and/or the presence of clonality in 100% of the cases included in our study; 90% of them harbored variants described to be valuable for diagnosis, prognosis or choice of treatment. In addition, the high depth of sequencing of the panel achieved detection of clones of minute size, and therefore allowed early detection of clonality, associated with potential relapse. Similarly, the possibility of following the dynamics of those genetic variants led us to identify persistent leukemia-associated mutations which are associated with a significant risk of relapse, and with reduced survival. Of note, NGS data detected clones harboring pathogenic variants in two patients with no MRD as per flow cytometry testing, indicating that NGS could complement the current gold standard follow-up method in some instances. Hence, NGS can help to improve the genetic characterization of AML and be complementary to current routine techniques for follow-up in AML patients.


The present study shows that our NGS panel has been useful for molecular diagnosis, monitoring of treatment efficacy, and early relapse detection in 90% of the AML cases included in our study, and useful for detection of clonality in 100% of them. Our NGS panel was also useful for following mutational clearance and/or clonal evolution in 63% of the total analyzed cases; specifically, it was of clinical utility in 79% of patients undergoing HSCT. Moreover, we could detect variants (and therefore clonality) in two cases who had tested negative on flow cytometry analysis. According to our data, NGS panels could be of clinical utility for routine follow-up in an elevated proportion of AML patients, as a complementary tool to immunophenotypic techniques for MRD monitoring.

Author’s Contributions

MJL collected data, analyzed data, contributed to PMP design and wrote the paper, AAD analyzed data and wrote the paper; IV collected data and contributed to PMP design; BA performed data analysis; AM, ZB and PA performed sample preparation and experiments; MCV, MTZ, EB, MCM, JRR, and AAP collected samples, diagnosed patients, and accrued patient clinical history; MFM wrote the paper and contributed to PMP design; FP and MJC conceived the project and contributed to PMP design. All authors reviewed the manuscript and approved its content.


This work was funded by the Government of Navarra, Department of Industry, Energy and Innovation (Project DIANA, 0011-1411-2017-000028). We particularly acknowledge the patients for their participation and the Biobank of the University of Navarra for its collaboration. MFM and MJC acknowledge funding from ISCIII (PI16/00159) and from the Spanish Association against Cancer (AECC, AIO2014). FP acknowledges funding from ISCIII (PI17/00701).


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