Acute Myeloid Leukemia - AML

Acute myeloid leukemias (AMLs) are a heterogeneous group of disorders characterized by the clonal expansion of myeloid blasts (eg, undifferentiated myeloid precursors) in the peripheral blood, bone marrow, and/or other tissues, which results in impaired hematopoiesis and bone marrow failure.     AML is the most common acute leukemia in adults; it accounts for approximately 80% of leukemia cases and the largest number of annual leukemia-related deaths in the United States.   The median age at diagnosis is 67 years, and 54% of patients are diagnosed at 65 years or older.  Advances in the treatment of AML have led to significant improvement in outcomes for younger patients; however, prognosis in the elderly, in whom the majority of new cases occur, remains poor.  

The initial laboratory evaluation for AML includes blood chemistry, coagulation studies, and a CBC with a differential of white blood cells (WBCs). In addition to morphologic evaluation of the bone marrow, immunophenotypic, cytogenetic, and molecular genetic ancillary studies are standard of care and are necessary for accurate classification, for risk stratification, and to guide therapy.

Quick Answers for Clinicians

What is the testing strategy for acute myeloid leukemia?

At diagnosis, the minimum acute myeloid leukemia (AML) workup includes a bone marrow aspirate for morphology, flow cytometric immunophenotyping, cytogenetics (eg, karyotyping with or without fluorescence in situ hybridization [FISH]), and appropriate molecular genetic testing.   

What is the strategy for minimal residual disease monitoring in acute myeloid leukemia?

Evaluation of minimal residual disease (MRD) in acute myeloid leukemia (AML) uses two main methodologies. Multiparametric flow cytometry is used to identify leukemia-associated immunophenotypes. Real-time quantitative polymerase chain reaction (qPCR)-based assays target specific leukemia-associated genetic abnormalities that are seen in a subset of patients.   

What role does next generation sequencing testing play in acute myeloid leukemia?

Next generation sequencing (NGS) has made it possible to look at a variety of mutations in multiple genes using a single assay; however, longer turnaround times sometimes limit the incorporation of this information into the routine workup for acute myeloid leukemia (AML). Sensitivity limitations of NGS-based assays currently limit their use in the assessment of minimal residual disease (MRD), although this is rapidly changing.   

Indications for Testing

Laboratory evaluation for suspected AML should be considered for individuals who present with signs of bone marrow infiltration (eg, anemia, thrombocytopenia), constitutional symptoms (eg, fever, fatigue, anorexia, weight loss), leukocytosis with circulating peripheral blasts, and/or AML-related complications (eg, tumor lysis syndrome, disseminated intravascular coagulation [DIC], leukostasis).   

Laboratory Testing

Diagnosis

The European Leukemia Net (ELN), the National Comprehensive Cancer Network (NCCN), the World Health Organization (WHO), and the European Society for Medical Oncology (ESMO) recommend morphologic assessment of the bone marrow for the initial workup for suspected AML.    Bone marrow aspirate and core biopsy enable bone marrow blasts to be enumerated and provide material for ancillary testing, including flow cytometric immunophenotyping, cytogenetics (eg, karyotyping with or without fluorescence in situ hybridization [FISH]), and molecular genetic testing (eg, quantitative polymerase chain reaction [qPCR] and/or next generation sequencing [NGS]). These studies allow for characterization, prognostication, treatment planning, and minimal residual disease (MRD) monitoring.    

Immunophenotyping​

In AML, flow cytometry is performed to detect leukemic blasts, determine blast lineage, and identify aberrant immunophenotypic features that enable the differentiation of abnormal blast populations from normal progenitors.   Flow cytometric immunophenotyping is frequently used in the assessment of MRD after chemotherapy.  

Cytogenetics

Cytogenetics are important in the diagnosis, prognosis, treatment, and monitoring of AML. In fact, pretreatment cytogenetic abnormalities constitute the single strongest prognostic factor for complete remission (CR) and overall survival (OS) in AML.   The WHO 2016 classification defines acute leukemia as ≥20% blasts in the marrow or peripheral blood; however, a diagnosis of AML may be made with <20% blasts in patients with certain recurrent cytogenetic abnormalities [eg, t(15;17), t(8;21), t(16;16), inv(16)].   

Cytogenetic testing methodologies include karyotyping and FISH. Karyotyping, also known as chromosomal analysis, is used to identify recurrent cytogenetic abnormalities. FISH is more sensitive than karyotyping for detecting potentially cryptic cytogenetic aberrations.

AML with Recurrent Genetic Abnormalities
Translocation Frequency Prognosis Predictive Factors

t(8;21)(q22;q22.1)

RUNX1-RUNX1T1

1-5% of AML cases

More common in younger patients

Usually associated with a high rate of CR and long-term, disease-free survival when treated with intensive consolidation therapy (eg, high-dose cytarabine)

Adverse

KIT mutations in adults

CD56 expression

inv(16)(p13.1;q22)

or

t(16;16)(p13.1;q22)

CBFB-MYH11

5-8% of cases in younger patients

Lower frequency in older adults

Usually associated with a high rate of CR and favorable OS when treated with intensive consolidation therapy (eg, high-dose cytarabine)

Improved

Chromosome 22 gain (as secondary abnormality)

Adverse

KIT mutations (especially involving exon 8) in adults

Older age

Increased WBCs​

FLT3 mutations (especially tyrosine kinase domain mutation, FLT-TKD)

Trisomy 8

Acute promyelocytic leukemia

t(15;17)(q22;q11-12)

PML-RARA

5-8% of cases in younger patients

Disease can occur at any age, but most patients are middle-aged adults

Lower frequency in older adults

Has more favorable prognosis when treated appropriately (with a combination of tretinoin and arsenic trioxide, with or without anthracycline for high-risk patients) than any other AML cytogenetic subtype

Adverse

Coagulopathy (associated with significant early death rates)

Hyperleukocytosis​

CD56 expression

FLT3-ITD mutation

Older age

Note: The significance of these features with current therapy is unclear

t(9;11)(p21.3;q23.3)

KMT2A-MLLT3

9-12% of AML cases in children

2% of AML cases in adults

Can occur at any age, but is more common in children

Generally intermediate survival, superior to that seen in AML with other 11q23.3 translocations

Note: Cases with t(9;11) and <20% blasts are not currently classified as AML (although this is controversial), but may be treated as such if clinically appropriate

Adverse

MECOM overexpression

t(6;9)(p23;q34.1)

DEK-NUP214

0.7-1.8% of AML cases

Occurs in both children and adults

Median pediatric patient age is 13 yrs; median age in younger adults is 35-44 yrs

Generally poor prognosis in both children and adults

Note: Cases with t(6;9)(p23;q34.1) and <20% blasts are not currently classified as AML (although this is controversial), but may be treated as such if clinically appropriate

Improved

Allogeneic hematopoietic stem cell transplant (may be associated with better OS)

Adverse

Increased WBCs (most predictive of shorter OS)

Increased bone marrow blasts (associated with shorter disease-free survival)

inv(3)(q21.3;q26.2)

or

t(3;3)(q21.3;q26.2)

GATA2, MECOM

1-2% of AML cases

Occurs most commonly in adults

No sex predilection

Aggressive disease with short survival

Outcome with <20% or >20% blasts is similarly poor

Cases with <20% blasts are not currently classified as AML (although this is controversial), but may be treated as such if clinically appropriate

Adverse

Complex karyotype and additional monosomy 7, regardless of blast percentage, are associated with a worse prognosis

Megakaryoblastic AML

t(1;22)(p13.3;q13.1)

RBM15-MKL1

Less than 1% of AML cases

Restricted to infants and young children (≤3 yrs); most cases occur in the first 6 mos of life (median patient age, 4 mos)

Uncommon abnormality in AML; when present, most commonly occurs in infants without trisomy 21

Female predominance

High-risk disease compared with pediatric acute megakaryocytic leukemia without t(1;22)

t(9;22)(q34.1;q11.2)

BCR-ABL1

<1% of AML cases

Occurs primarily in adults

Possible male predominance

Aggressive disease with poor response to traditional AML therapy or TKI therapy alone

Improved

TKI therapy followed by allogeneic hematopoietic cell transplant

AML-MRC

AML-MRC

24-35% of all AML cases

Occurs primarily in the elderly

Rare in children

Generally has a poor prognosis with a lower rate of CR than other AML subtypes

Improved

Previous MDS cases with relatively low blast counts may constitute less clinically aggressive disease

Previous MDS cases with 20-29% bone marrow blasts may behave in a manner more similar to that of MDS than other AMLs

Adverse

In the absence of a CEBPA double mutation or NPM1 mutation, multilineage dysplasia still appears to confer a poor prognosis, although not as poor as the prognosis for cases with high-risk cytogenetic abnormalities

ASXL1 mutations may be associated with a worse prognosis

CD14 expression

CD11b expression

t-MNs

t-MNs

10-20% of all cases of AML, MDS, and MDS/MPN​

Most patients have been treated for a previous malignancy

70% of patients have been treated for a solid tumor (breast cancer most common)

30% of patients have been treated for a hematologic neoplasm (non-Hodgkin lymphoma is most common)

5-20% of t-MN cases occur after therapy for a nonneoplastic disorder

Poor

5-yr survival rates (<10%)

Improved

Cases with balanced chromosomal translocations generally have a better prognosis; however, such cases, except those with t(15;17) and inv(16) or t(16;16), have shorter median survival times than their de novo counterparts

Adverse

Cases associated with abnormalities in chromosomes 5 and/or 7, TP53 mutations, and a complex karyotype have a particularly poor outcome, with a median survival time of <1 yr

AML-MRC, AML with myelodysplasia-related changes; ITD, internal tandem duplication; MDS, myelodysplastic syndrome; MPN, myeloproliferative neoplasm; TKD, tyrosine kinase domain; TKI, tyrosine kinase inhibitor; t-MNs, therapy-related myeloid neoplasms

Source: WHO, 2017 

Molecular Genetics

Gene alterations, along with translocations and inversions, carry prognostic importance in AML. In addition to large chromosomal rearrangements, molecular changes have also been implicated in the development of AML. In fact, genetic mutations are identified in more than 97% of cases, often in the absence of any large chromosomal abnormality.  A comprehensive evaluation of several molecular markers (eg, FLT3, NPM1, CEBPA, KIT, IDH1, and IDH2) is important for risk assessment and prognostication in certain patients with AML and may guide treatment decisions.  

AML with Gene Mutations
Molecular Genetic Alteration Frequency Cytogenetic Group Prognostic Significance
NPM1 mutationa

2-8% of cases in children

27-35% of cases in adults

45-64% of adult cases with normal karyotype

Normal karyotype

Favorable prognosis [comparable to that of AML with t(8;21) or AML with inv(16) or t(16;16)] in patients with normal karyotype and without FLT3-ITD mutation

Poorer prognosis in young adults with FLT3-ITD mutation, but more favorable prognosis than wild-type NPM1 with FLT3-ITD mutation

Poor prognosis if present with FLT3-ITD and DNMT3A mutations

Biallelic CEBPA mutationsa 4-9% of cases in children and young adults Normal karyotype Favorable prognosis, similar to that of AML with inv(16) or t(8;21)
Single CEBPA mutations Normal karyotype Poor prognosis compared to biallelic CEBPA mutations
RUNX1 mutationa,b 4-16% of AML cases Normal karyotype Poor prognosis; poorer prognosis if present with ASXL1 mutation
KIT mutation 4% of AML cases t(8;21)(q22;q22.1)

Poor prognosis

Increased risk of relapse

FLT3-ITD 28% of AML cases Normal karyotype Poor prognosis
WT1 mutation Normal karyotype Poor prognosis
TET2 mutation Normal karyotype Poor prognosis
ASXL1 mutation Normal karyotype Poor prognosis
DNMT3A mutation Normal karyotype Poor prognosis
TP53 alterations (mutation or loss) 2-8% of AML cases Complex karyotype (≥3 abnormalities) Poor prognosis

aClassified as a provisional entity by WHO.

bAssociated with radiation exposure and alkylating agent chemotherapy.

Sources: Weinberg, 2017 ; De Kouchkovsky, 2016 ; Arber, 2016 

Monitoring

Monitoring relies on knowledge of the leukemia-associated phenotype defined at diagnosis for the detection of MRD. The most frequently used methods for monitoring include flow cytometry for abnormal immunophenotypes and PCR-based assays to detect previously identified mutations. 

ARUP Laboratory Tests

Immunophenotyping

Flow Cytometry

Use for diagnosis and monitoring

Specimen: bone marrow, whole blood, tissue, fluid

Cytogenetics

Karyotyping

Use for diagnosis, prognosis, and monitoring

Specimen: bone marrow

Specimen: bone marrow

Specimen: leukemic blood

FISH

Use for risk stratification and therapeutic management in newly diagnosed AML

Use if FISH probes other than those included in the standard panels below are desired

Specimen: bone marrow, whole blood

Use to identify important prognostic abnormalities in newly diagnosed AML

Specimen: bone marrow, whole blood

Probes include t(15;17)(q24;q21) PML-RARA; t(8;21)(q22;q22) RUNX1T1-RUNX1 (ETO-AML1); inv(16)(p13.3q22) CBFB; 11q23 KMT2A (MLL); inv(3) or t(3;3) RPN1-MECOM (EVI1); del(5)(q31) EGR1; and del(7)(q31)/-7 D7S486

Use in conjunction with conventional cytogenetics for diagnosis, prognosis, and monitoring of MRD in therapy-related MDS or AML associated with MDS

Specimen: bone marrow, whole blood

Probes include -5/del(5q); -7/del(7q); and 11q23 rearrangements (targets MLL)

Use for rapid genetic confirmation of acute promyelocytic leukemia

Specimen: bone marrow, whole blood (when hyperleukocytosis is present and bone marrow aspiration is not possible)

PML probe targets the PML gene (15q22); RARA probe targets the RARA gene (17q21.1)

Molecular Testing

PCR

Use to detect and quantitate PML-RARA translocation arising from t(15;17)

Use to monitor for MRD and assess the risk of disease relapse

Specimen: bone marrow (preferred for maximum sensitivity), whole blood

Use to detect and quantitate CBFB-MYH11 inv(16) fusion transcripts

Use to monitor for MRD and assess the risk of disease relapse

Specimen: bone marrow (preferred for maximum sensitivity), whole blood

Use to detect and quantitate RUNX1-RUNX1T1 fusions arising from t(8;21)

Use to monitor for MRD and assess the risk of disease relapse

Specimen: bone marrow (preferred for maximum sensitivity), whole blood

Use to detect and quantitate NPM1 mutant transcripts (type A, B, and D)

Use to monitor for MRD and assess the risk of disease relapse

Specimen: bone marrow, whole blood

Initial test for prognostication of cytogenetically normal (CN)-AML

Aids in the diagnosis and management of AML

Not intended for MRD monitoring

Use to detect IDH1 and IDH2 mutations in whole blood or bone marrow

Targeted therapies are available for these mutations

Provides diagnostic, prognostic, and predictive information for AML associated with inv(16) or t(8;21) (also known as core binding factor [CBF] AML)

Next Generation Sequencing

Use to assess for single gene mutations, including substitutions and smaller insertions and deletions, that may have prognostic and/or therapeutic significance in AML

3002714

Specimen: bone marrow, whole blood

Components: ANKRD26, ASXL1, CEBPA, DDX41, DNMT3A, ETV6, FLT3, GATA2, IDH1, IDH2, KIT, KRAS, NPM1, NRAS, RUNX1, TP53, WT1

Use to assess for somatic variants in genes associated with myeloid malignancies that are relevant for diagnosis, prognosis, or clinical management in patients with AML and related disorders

Specimen: bone marrow, whole blood

Components: ANKRD26, ASXL1, ASXL2, BCOR, BCORL1, BRAF, CALR, CBL, CBLB, CEBPA, CSF3R, CUX1, DDX41, DNMT1, DNMT3A, EED, ELANE, ETNK1, ETV6, EZH2, FAM5C, FBXW7, FLT3, GATA1, GATA2, GNAS, HNRNPK, IDH1, IDH2, IL7R, JAK1, JAK2, JAK3, KDM6A, KIT, KMT2A, KRAS, LUC7L2, MAP2K1, MLL, MPL, NOTCH1, NPM1, NRAS, NSD1, PHF6, PIGA, PRPF40B, PRPF8, PTPN11, RAD21, RUNX1, SETBP1, SF1, SF3A1, SF3B1, SH2B3, SMC1A, SMC3,SRSF2, STAG2, STAT3, STAT5B, SUZ12, TET2, TP53, U2AF1, U2AF2, WT1, ZRSR2 (one or more exons of the preferred transcript are not covered by sequencing for CUX1, DNMT1, KDM6A, NPM1, STAT5B, SUZ12)

Use to assess for inherited/germline DNA variants associated with familial myeloid dysplasias and malignancies

Specimen: cultured skin fibroblasts (preferred), whole blood, skin punch biopsy

Components: ANKRD26, ATM, BLM, CBL, CEBPA, DDX41, ELANE, ETV6, GATA1, GATA2, KRAS, NBN, PTPN11, RUNX1, SAMD9, SAMD9L, SRP72, TERC, TERT, TP53

Medical Experts

Contributor

Karner

Kristin Hunt Karner, MD
Assistant Professor of Pathology (Clinical), University of Utah
Medical Director, Hematopathology and Molecular Oncology, ARUP Laboratories
Contributor

March

Jordon K. March, MD
Jordon K. March, MD
Anatomic and Clinical Pathology Resident, University of Utah School of Medicine and ARUP Laboratories
Associate Medical Director, ARUP Consult

References

  1. 32171751

    Heuser M, Ofran Y, Boissel N, et, al. a href="https://pubmed.ncbi.nlm.nih.gov/32171751/">Acute myeloid leukaemia in adult patients: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2020;S0923-7534(20)36079-8.

    PubMed
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