Diagnostics

Acute myeloid
leukemia (AML)

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Cytomorphology
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Immunophenotyping
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Chromosome analysis
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Molecular genetics
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Classification
Diagnostics
MRD
Prognosis
Therapy
Recommendation

Based on the current guidelines and the current state of research, there are different diagnostic recommendations for patients with acute myeloid leukemia. We have summarized the most important information on classification and diagnostic methods at MLL. In addition, we provide further links on prognosis and therapy in acute myeloid leukemia, so that you can inform yourself in more detail.

AML: Classification

Acute myeloid leukemias (AML) may arise de novo, after prior cytotoxic and/or radiotherapy (AML-pCT), or secondarily from preexisting myelodysplastic/myeloproliferative disease or MDS. In the current classification, the WHO distinguishes between AML with defining genetic abnormalities and AML, defined by differentiation (Table 1) as well as myeloid sarcoma (WHO 2022).

Tab. 1: AML with defining genetic abnormalities and AML, defined by differentiation (WHO 2022)

AML with defining genetic abnormalities	AML, defined by differentiation
Acute promyelocytic leukemia with PML::RARA fusion	AML with minimal differentiation
AML with RUNX1::RUNX1T1 fusion	AML without maturation
AML with CBFB::MYH11 fusion	AML with maturation
AML with DEK::NUP214 fusion	Acute basophilic leukemia
AML with RBM15::MRTFA fusion	Acute myelomonocytic leukemia
AML with BCR::ABL1 fusion	Acute monocytic leukemia
AML with KMT2A rearrangement	Acute erythroid leukemia
AML with MECOM rearrangement	Acute megakaryoblastic leukemia
AML with NUP98 rearrangement
AML with NPM1 mutation
AML with CEBPA mutation
AML, myelodysplasia-related (AML-MR)
AML with other defined genetic alterations

In contrast to the previous 2017 version of the WHO classification, the previous criterion of at least 20% blasts in peripheral blood or bone marrow (Swerdlow et al. 2017) is omitted for AML with defining genetic abnormalities – with the exception of AML with BCR::ABL1 fusion, AML with CEBPA mutation and AML-MR. For AML with BCR::ABL1 fusion, this serves to differentiate it from CML. For the diagnosis of AML with CEBPA mutation, the blast criterion must also still be met due to insufficient data to date (WHO 2022).

Important changes in the new WHO classification also concern the AML-MR subtype (previously AML with myelodysplasia-related changes). Essential diagnostic criteria include a blast count of at least 20% in peripheral blood or bone marrow. However, for this subtype, morphology is no longer the sole diagnostic criterion, cytogenetic criteria have been updated, and a mutation-based definition has been introduced using 8 genes:

Defining cytogenetic abnormalities in AML-MR:

Complex karyotype (≥ 3 abnormalities)
5q deletion or loss of 5q due to unbalanced translocation
Monosomy 7, 7q deletion, or loss of 7q due to unbalanced translocation
11q deletion
12p deletion or loss of 12p due to unbalanced translocation
Monosomy 13 or 13q deletion
17p deletion or loss of 17p due to unbalanced translocation
Isochromosome 17q
idic(X)(q13)

Defining somatic mutations in AML-MR:

ASXL1
BCOR
EZH2
SF3B1
SRSF2
STAG2
U2AF1
ZRSR2

Genetic changes are also becoming increasingly important in pediatric AML (pAML): Umeda et al. published a new genomic classification for categorizing pAML (Umeda et al. 2024).

AML: Diagnostic methods and their relevance

Cytomorphology together with cytochemistry (myeloperoxidase and esterase) is part of standard diagnostics. It serves to confirm the diagnosis and is also required for the classification of AML, defined by differentiation according to WHO. Cytomorphological remission assessment is still a gold standard for follow-up during therapy.

Immunophenotyping is always part of the routine diagnosis of AML. Important markers here are HLA-DR and CD34 in acute promyelocytic leukemia (APL), CD19 and CD56 in AML with maturation and AML with RUNX1::RUNX1T1 fusion, and CD2, CD15, and CD34 in myelomonocytic AML with abnormal eosinophils. In AML with minimal differentiation, CD13, CD33, and CD117, among others, play an important role. In addition, differentiation from ALL is significant here. Lymphoid antigens are not expressed or the lymphoid score is not sufficient for the diagnosis of biphenotypic acute leukemia. In approximately half of cases, despite cytochemical negativity for myeloperoxidase, expression of MPO can be detected by flow cytometry, leading to a diagnosis of AML. Also, acute megakaryoblastic leukemia is negative for MPO and esterase by cytochemistry. Due to the frequent presence of myelofibrosis, cytologic evaluation is difficult. Immunophenotyping demonstrates expression of CD41 or CD61.

Classical chromosome analysis to determine the karyotype of the leukemia cells is now part of standard diagnostics for every AML patient. The karyotype and the numerous characteristic chromosomal aberrations are used for classification according to WHO and represent the most important independent prognostic parameters. For some genetically defined subtypes of AML, therapeutic consequences are derived from the findings of chromosome analysis.

Tab. 2: Recurrent chromosomal aberrations in AML (Grimwade et al. 2001, Grimwade et al. 2010, Grimwade et al. 2016, Döhner et al. 2022)

Cytogenetics	Genes	Frequency*
t(15;17)(q24;q21)	PML::RARA	2-13%
t(8;21)(q22;q22)	RUNX1::RUNX1T1	2-7%
inv(16)(p13q22) / t(16;16)(p13;q22)	CBFB::MYH11	1-5%
11q23	KMT2A (MLL-X)	1-4%
t(9;22)(q34;q11)	BCR::ABL1	1%
t(6;9)(p23;q34)	DEK::NUP214	1%
t(5;11)(q35;p15)	NUP98::NSD1	1%
inv(3)(q21q26)	GATA2::MECOM (EVI1)	1%
t(3;21)(q26;q22)	RUNX1::MECOM (EVI1)	<1%
t(7;11)(p15;p15)	NUP98::HOXA9	<1%
t(8;16)(p11;p13)	KAT6A::CREBBP	<1%
t(16;21)(p11;q22)	FUS::ERG	<1%
t(3;5)(q25;q35)	NPM1::MLF1	<1%
t(10;11)(p13;q21)	PICALM::MLLT10	<1%
5q31 deletion	CDC25C, EGR1	2-10%
5q33 deletion	RPS14	2-10%
7q31 deletion/-7		2-5%/5-9%
17p13 deletion	TP53	n/a

*Frequencies may vary by age group, compiled from Grimwade et al. 2001, 2010 and 2016.

FISH analysis serves as an important complement to classical chromosome analysis and answers specific questions, e.g. the detection of the translocation t(15;17)(q24;q21) – PML::RARA in suspected APL. A PML::RARA rearrangement can be detected in as few as 3 hours. Common unbalanced changes, such as deletions of the long arm of chromosome 5 or 7, monosomies (-7) or trisomies (+8, +11, +13, +21) can also be detected with FISH. With the help of a reasonable FISH probe set, even a large proportion of AML cases with complex aberrant karyotype can be correctly classified.

Chromosome painting with 1-3 or 24 colors (24-color FISH) on metaphase chromosomes can be used for clarification in cases of ambiguous classical chromosome analysis, which often occurs in complex aberrant karyotypes.

Today, the molecular genetic diagnosis of AML is driven by next-generation sequencing (NGS). In this way, extensive panels can keep the examination time as short as possible and generate a comprehensive picture of the molecular landscape with diagnostic, prognostic and therapeutic significance. However, conventional PCR for the detection of fusion genes, fragment length analysis ("gene scan") for FLT3-ITD, and quantitative real-time PCR (KMT2A-PTD, fusion genes, EVI1 expression) are also diagnostic methods. Recurrent mutations in AML are shown in Table 3.

The "AML-ELN Panel according to Döhner et al. Blood 2022" focuses on the genes recommended for testing at diagnosis and can be complemented by the detection of the fusion transcripts BCR::ABL1, CBFB::MYH11, DEK::NUP214, PML::RARA and RUNX1::RUNX1T1. In contrast, the "AML/targeted therapy" panel aims to identify potential candidates for targeted therapy by mutational analysis of FLT3 (TKD and ITD) and IDH1/2.

Tab. 3: Molecular mutations in AML (Grimwade et al. 2016, Yu et al. 2020)

Mutation	Frequency in cytogenetic subgroup	Total frequency
FLT3-ITD		~25%
FLT3-TKD		~7-10%
NPM1	normal karyotype: 40-60%	~30%
CEBPA	normal karyotype: 10-20% CEBPA^bi: ~50%	10-20%
KMT2A-PTD	more frequent with normal cytogenetics and +11	3.2-11%
IDH1/2		~10%
DNMT3A		13.5-23%
KIT	t(8;21): ~25% inv(16): ~35%
NRAS	inv(16): ~40% 11q23: ~20% inv(3): ~40%	11%
KRAS	inv(16): ~10% 11q23: ~20%	5-11%
ASXL1		~15-20%
TP53	s-AML/AML-pCT: 20-37% complex karyotype: ~70%	<10%

AML: MRD (Measurable Residual Disease)

Determination of MRD in AML serves as a prognostic/predictive biomarker for refined risk stratification and treatment decisions, as a monitoring tool for early detection of relapse, and as a potential endpoint in clinical trials. Highly sensitive quantitative detection methods (sensitivity 0.01-0.001%) are available for a variety of molecular genetic markers or fusion transcripts that are used to assess recurrence risk and eligibility for allogeneic stem cell transplantation. For example, for patients with mutated NPM1, RUNX1::RUNX1T1 fusion, CBFB::MYH11 fusion, or PML::RARA fusion, MRD determination by PCR is recommended (Heuser et al. 2021). MRD negativity represents a very favorable parameter for survival and low risk of recurrence (Schuurhuis et al. 2018, Freeman & Hourigan 2019, Rücker et al. 2019). Moreover, immunophenotyping (sensitivity 0.01%) now plays an increasingly important role in MRD determination and therapy stratification (Schuurhuis et al. 2018, Heuser et al. 2021). The FISH method can also be used in case of a positive marker, which cannot be monitored by PCR or LAIP. However, it is less sensitive (1-5%). NGS-based MRD diagnostics may also be useful for prognostic assessment. A detailed review on MRD in AML is available in European LeukemiaNet (ELN) guidelines by Heuser et al. and Döhner et al. (Heuser et al. 2021, Döhner et al. 2022) as well as in a recent review by Chea et al. (Chea et al. 2024).

AML: Prognosis

In addition to age, leukocyte count, and general health, karyotype and molecular genetic alterations represent important prognostic parameters and have a major impact on the therapeutic strategy. Modern genetic prognostic systems combine molecular mutations and cytogenetics (Grimwade et al. 2016, Döhner et al. 2022). An overview of the risk groups according to the European LeukemiaNet (ELN) classification can be found in Table 4.

Tab. 4: Genetic risk classification at initial diagnosis according to ELN^a (Döhner et al. 2022)

Risk Category	Cyto- and molecular genetic abnormality
Favorable	t(8;21)(q22;q22.1); RUNX1-RUNX1T1^b,c inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11^b,c Mutated NPM1^b,dwithout FLT3-ITD bZIP in-frame mutated CEBPA^e
Intermediate	Mutated NPM1^b,d with FLT3-ITD Wild-type NPM1 with FLT3-ITD t(9;11)(p21.3;q23.3); KMT2A:: MLLT3^b,f Cytogenetic and/or molecular abnormalities not classified as favorable or adverse
Adverse	t(6;9)(p23;q34.1); DEK::NUP214 t(v;11q23.3); KMT2A-rearranged (exclusive KMT2A-PTD) t(9;22)(q34.1;q11.2); BCR::ABL1 t(8;16)(p11;p13)/KAT6A::CREBBP inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2); GATA2::MECOM (EVI1) t(3q26.2;v)/MECOM(EVI1)-rearranged -5 or del(5q); -7; -17/abnl(17p) Complex karyotype^g, monosomal karyotype^h Mutated ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 or ZRSR2ⁱ Mutated TP53^j

^a Frequencies, response rates and outcomes should be reported by risk category and, if sufficient, by specific genetic lesion.

^b Mainly based on outcomes observed in intensively treated patients. Initial risk categorization may change over the course of treatment based on MRD measurements.

^c Simultaneous presence of KIT and/or FLT3 mutations does not change the risk classification.

^dAML with NPM1 mutation and cytogenetic abnormalities with unfavorable risk are categorized as unfavorable risk.

^e The favorable prognosis for CEBPA mutation applies regardless of whether it is a monoallelic or biallelic mutation, as long as it is an in-frame mutation of the bZIP region.

^f The presence of t(9;11)(p21.3;q23.3) takes precedence over rare, concomitant gene mutations with unfavorable risk.

^g ≥ 3 independent aberrations in the absence of WHO-defined recurrent chromosomal aberrations; excludes hyperdiploid karyotypes with three or more trisomies (or polysomies) without structural abnormalities.

^h two or more monosomies (except loss of the X or Y chromosome) or single monosomy in combination with at least one structural aberration (except in core-binding-factor (CBF) AML).

ⁱ currently, these markers should not be classified as unfavorable prognostic markers if they occur together with AML subtypes of the favorable risk group.

^j TP53 mutation with a proportion of variant alleles of at least 10%, regardless of TP53 allele status (mono- or biallelic mutation); TP53 mutations are significantly associated with AML with complex and monosomal karyotypes.

AML: Therapy

Therapy options and algorithms can be found from the European LeukemiaNet (Döhner et al. 2022), the American Society of Hematology (Sekeres et al. 2020), the European Society of Medical Oncology (Heuser et al. 2020), and the Onkopedia website (Onkopedia Guideline AML) as well as from the National Comprehensive Cancer Center, among others.

Chea M et al. Minimal Residual Disease in Acute Myeloid Leukemia: Old and New Concepts. Int J Mol Sci 2024;25(4):2150.

Döhner H et al. Diagnosis and Management of AML in Adults: 2022 ELN Recommendations from an International Expert Panel. Blood 2022;

Freeman SD, Hourigan CS. MRD evaluation of AML in clinical practice: are we there yet? Hematology Am Soc Hematol Educ Program 2019;2019(1):557-569.

Grimwade D et al. The predictive value of hierarchical cytogenetic classification in older adults with acute myeloid leukemia (AML): analysis of 1065 patients entered into the United Kingdom Medical Research Council AML11 trial. Blood 2001;98(5):1312-1320.

Grimwade D et al. Refinement of cytogenetic classification in acute myeloid leukemia: determination of prognostic significance of rare recurring chromosomal abnormalities among 5876 younger adult patients treated in the United Kingdom Medical Research Council trials. Blood 2010;116(3):354-365.

Grimwade D et al. Molecular landscape of acute myeloid leukemia in younger adults and its clinical relevance. Blood 2016;127(1):29-41.

Heuser M et al. 2021 Update on MRD in acute myeloid leukemia: a consensus document from the European LeukemiaNet MRD Working Party. Blood 2021;138(26):2753-2767.

Heuser M et al. Acute myeloid leukaemia in adult patients: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2020;31(6):697-712.

Onkopedia Leitlinie „Akute myeloische Leukämie (AML)“ 2023; DGHO 2023.

Rücker FG et al. Measurable residual disease monitoring in acute myeloid leukemia with t(8;21)(q22;q22.1): results from the AML Study Group. Blood 2019;134(19):1608-1618.

Schuurhuis GJ et al. Minimal/measurable residual disease in AML: a consensus document from the European LeukemiaNet MRD Working Party. Blood 2018;131(12):1275-1291.

Sekeres MA et al. American Society of Hematology 2020 guidelines for treating newly diagnosed acute myeloid leukemia in older adults. Blood Adv 2020;4(15):3528-3549.

Swerdlow SH et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. International Agency for Research on Cancer (IARC) 2017; 4th.

Taube F et al. CEBPA mutations in 4708 patients with acute myeloid leukemia: differential impact of bZIP and TAD mutations on outcome. Blood 2022;139(1):87-103.

Umeda M et al. A new genomic framework to categorize pediatric acute myeloid leukemia. Nat Genet 2024;56(2):281-293.

WHO Classification of Tumours Editorial Board. Haematolymphoid tumours [Internet; beta version ahead of print]. Lyon (France): International Agency for Research on Cancer; 2022. (WHO classification of tumours series, 5th ed.; vol. 11). Available from: https://tumourclassification.iarc.who.int/chapters/63.

Yu J et al. Clinical implications of recurrent gene mutations in acute myeloid leukemia. Exp Hematol Oncol 2020;9(4).

Status: June 2024

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Acute myeloid leukemia (AML)

AML: Classification

Tab. 1: AML with defining genetic abnormalities and AML, defined by differentiation (WHO 2022)

AML: Diagnostic methods and their relevance

Cytomorphology

Immunophenotyping

Chromosome analysis

Tab. 2: Recurrent chromosomal aberrations in AML (Grimwade et al. 2001, Grimwade et al. 2010, Grimwade et al. 2016, Döhner et al. 2022)

Fluorescence in situ hybridisation (FISH)

Molecular genetics

Tab. 3: Molecular mutations in AML (Grimwade et al. 2016, Yu et al. 2020)

AML: MRD (Measurable Residual Disease)

AML: Prognosis

Tab. 4: Genetic risk classification at initial diagnosis according to ELNa (Döhner et al. 2022)

AML: Therapy

You may also be interested in

Acute myeloid
leukemia (AML)

Tab. 4: Genetic risk classification at initial diagnosis according to ELN^a (Döhner et al. 2022)