Acute Myeloid Leukemia - AML

Primary Author: Kelley, Todd, MD, MS.

  • Key Points
  • Diagnosis
  • Monitoring
  • Background
  • Lab Tests
  • References
  • Related Content

Two challenging areas in the evaluation of acute myeloid leukemia (AML) include the diagnosis of acute promyelocytic leukemia (APL) and the prognostication of cytogenetically normal AML (CN-AML).

Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is a rare subtype of AML that is associated with fatal coagulopathies and hemorrhage. Rapid diagnosis is imperative for reducing morbidity/mortality. APL is readily treatable with all-trans retinoic acid (ATRA), a distinctively different agent from most drugs used in AML. Treatment may be initiated before genetic confirmation is completed; however, genetic confirmation of PML-RARA fusion is mandatory in all cases.

Cytogenetically Normal AML (CN-AML) Prognostic Markers

Cytogenetic analysis is the initial test for risk stratification in acute myeloid leukemia (AML). Fifty percent of patients with AML are cytogenetically normal (CN-AML) and considered to be at intermediate risk, but single gene mutations exist that provide subtype stratification within this risk group. Use of these molecular markers in CN-AML may provide better prognostication and aid in determination of therapeutic regimens. For prognostication purposes, all markers should be interpreted as a group and not individually.

Extracted DNA/RNA can be stored from original specimen and used for additional testing after determination of initial cytogenetic risk classification.

Indications for Testing

  • Anemia, thrombocytopenia, and/or blasts (with or without Auer rods) on peripheral smear

Laboratory Testing

  • Initial testing should include CBC with platelet count and peripheral smear to identify blasts
  • Prothrombin time, partial thromboplastin time and fibrinogen if thrombocytopenia is present (typically promyelocytic leukemias)
    • D-dimer if DIC is suspected

Histology

  • Combination of methods for diagnosis used includes morphology, immunohistochemistry, cytogenetics, and flow cytometric immunophenotyping
    • Key distinction is between AML and acute lymphoblastic leukemia (ALL)
      • AML will demonstrate predominantly myeloid markers and sufficient numbers of lymphoid antigens to raise the possibility of mixed-phenotype acute leukemia
    • Bone marrow biopsy with chromosome analysis
      • Diagnosis of AML requires >20% blasts in peripheral blood or bone marrow unless a recurrent cytogenetic abnormality is present to allow diagnosis in the presence of fewer blasts (eg, CBFB-MYH11)
    • Flow cytometry – establishes myeloid or monocytic lineage
      • Monocytic – CD36, 64, 14, 33+
      • Myeloid – CD13, 15, 33, MPO+
      • Megakaryocytic – CD36+, 41+, 61+
      • Erythroid – CD36, 71+, GlyA+
    • Cytogenetics – provides genetic classification of leukemia; aids in decisions regarding post-inductive therapies
      • Usually assessed using PCR, SNP microarray, next generation sequencing, and/or FISH testing
      • SNP microarray cannot detect t(15;17) (PML-RARA), t(8;21) (RUNX1T1-RUNX1), or balanced rearrangement of MLL or CBFB gene
    • Immunohistochemistry
      • Consider staining for CD19, CD34, CD68, CD79a, CD117, lysozyme, myeloperoxidase, and Pax-5

Prognosis

  • Dependent on karyotyping, type of AML, and cytogenetic results
  • Type of AML
    • Favorable
      • Promyelocytic
      • Megakaryocytic in Down syndrome
  • Differential Diagnosis

    • Bone marrow biopsy with PCR, FISH, and/or chromosome analysis every 3 months
      • Morphology and immunophenotypic (flow cytometry) bone marrow examination may be done more frequently to assess response to therapy
    • Minimal residual disease (MRD)
      • Chromosome FISH interphase testing – probe detection limit is ~2-5%
      • Depending on clinical setting, flow cytometry or PCR test may be informative
      • Cannot use FISH for PML-RARA due to inadequate sensitivity
      • Immunophenotyping

    Acute myeloid leukemia (AML) is a malignant neoplasm of hematopoietic bone marrow precursor cells and is the most common type of acute leukemia in adults. Acute leukemias phenotypically represent immature hematopoietic cells but often display differences from normal cell counterparts.

    Epidemiology

    • Incidence
      • Adults – ~3/100,000
      • Children – 0.7/100,000
      • Acute promyelocytic leukemia (APL) – 0.23/100,000
    • Age – more common in elderly; median age of 67 years at diagnosis (NCCN, 2015)
    • Sex – M>F (minimal)

    Current Classification

    • Based on blast cytogenetic features, differentiation, and morphology
    • Genetic studies are important in classification and prognosis

    Risk Factors

    • Myeloproliferative neoplasm (MPN) or myelodysplastic syndrome (MDS) – chronic myelogenous leukemia, polycythemia vera, essential thrombocythemia, idiopathic myelofibrosis
    • Environmental risk factors – ionizing radiation, benzene, petrochemicals, pesticides
    • Genetic – Down syndromeFanconi anemiaBloom syndrome, Wiskott-Aldrich syndrome, sibling with AML
    • Therapy-related leukemia – previous cytotoxic chemotherapy (alkylating agents, topoisomerase II inhibitors)

    Pathophysiology

    • Abnormal proliferation of myeloid precursor cells characterized by
      • Decreased rate of cell self-destruction
      • Arrest of cellular differentiation
    • Leukemic cells have survival advantage
      • Leukemic cells infiltrate bone marrow
      • Blasts and immature cells may populate peripheral blood; however, some patients present with leukopenia

    Clinical Presentation

    • Nonspecific symptoms often due to bone marrow infiltration
      • Anemia – weakness, pallor
      • Thrombocytopenia – bruising
    • Leukocytosis with increased peripheral blood blasts
    • Thrombocytopenia, anemia
    • Myeloid sarcoma – mass lesion of leukemic cells in tissue
      • Also known as chloroma or extramedullary myeloid tumor
    • Complications

    Indications for Laboratory Testing

    Tests generally appear in the order most useful for common clinical situations.
    Click on number for test-specific information in the ARUP Laboratory Test Directory

    Chromosome Analysis, Bone Marrow with Reflex to Genomic Microarray 2007130
    Method: Giemsa Band/Genomic Microarray (Oligo-SNP array)

    Follow Up

    Repeat testing as clinically indicated to monitor disease progression

    Leukemia/Lymphoma Phenotyping by Flow Cytometry 2008003
    Method: Flow Cytometry

    Limitations

    Some hematopoietic neoplasms do not show phenotypic abnormalities and therefore may not be detected by flow cytometry

    Poor cell viability may adversely affect antigens and impede the ability to properly identify neoplastic cells

    Flow results cannot be used alone to diagnose malignancy; should be interpreted in conjunction with morphology, clinical information, and other necessary ancillary tests for a definitive diagnosis

    Cytogenomic SNP Microarray - Oncology 2006325
    Method: Genomic Microarray (Oligo-SNP Array)

    Limitations

    Low-level mosaicism (<15% to 20%) may not be detected; test may not be appropriate for individuals with expected lower levels of malignant cells

    Does not detect balanced rearrangements; FISH should be used to evaluate specific balanced rearrangements according to indication

    Does not detect base pair mutations and very small deletions/duplications; imbalances of the mitochondrial genome; positional information for chromosome rearrangements, low level clones

    Not recommended for MRD

    Myeloid Malignancies Somatic Mutation and Copy Number Analysis Panel 2012182
    Method: Massively Parallel Sequencing/Genomic Microarray (Oligo-SNP Array)

    Limitations

    Mutations may be present below the limit of detection

    Not intended to detect minimal residual disease (MRD)

    Myeloid Malignancies Mutation Panel by Next Generation Sequencing 2011117
    Method: Massively Parallel Sequencing

    Limitations

    Mutations may not be present below the limit of detection

    Not intended to detect MRD

    Chromosome FISH, Interphase 2002298
    Method: Fluorescence in situ Hybridization

    Limitations

    Limit of detection is probe dependent and ~2-5% in interphase nuclei; residual disease levels lower than this will likely appear normal

    Some of these abnormalities can also be detected in myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPN) and therefore are not by themselves sufficient for diagnosis but rather consistent with the suspected diagnosis

    Follow Up

    Repeat testing as clinically indicated to monitor disease progression

    Acute Myeloid Leukemia Panel by FISH 2011132
    Method: Fluorescence in situ Hybridization

    Limitations

    Chromosome alterations not targeted by the panel probes will not be detected

    Acute Myelogenous Leukemia (AML) with Myelodysplastic Syndrome (MDS) or Therapy-Related AML, by FISH 2002653
    Method: Fluorescence in situ Hybridization

    Limitations

    MLL gene at 11q23 has multiple translocation partners which are not identified by this test

    Panel detects only the specific aberrations targeted by the probes

    CBFB-MYH11 inv(16) Detection, Quantitative 2011114
    Method: Reverse Transcription Quantitative Polymerase Chain Reaction

    Limitations

    Limit of detection (LOD) for types A, D is one copy

    LOD for CBFB-MYH11 type E is 10 copies

    Presence of fusion product <10 copies may not be detected

    Bone marrow specimens preferred for maximum sensitivity

    Poor RNA yield will lead to false negatives

    RUNX1-RUNX1T1 (AML1-ETO) t(8;21) Detection, Quantitative 2010138
    Method: Quantitative Reverse Transcription Polymerase Chain Reaction

    Limitations

    “Not Detected” does not exclude the possibility of RUNX1-RUNX1T1 transcripts below the test limit of detection

    BM samples preferred for maximum sensitivity

    Poor RNA yield and/or quality due to sample age or hypocellularity will negatively impact the test

    NPM1 Mutation by PCR and Fragment Analysis 0040174
    Method: Polymerase Chain Reaction/Fragment Analysis

    Limitations

    Negative test result does not exclude presence of mutations in transcripts below the detection limit, presence of rare mutations not detected by this test

    All markers should be interpreted as a group and not individually

    CEBPA Mutation Detection 2004247
    Method: Polymerase Chain Reaction/Sequencing

    Limitations

    Negative test result does not exclude presence of mutations in transcripts below the detection limit, presence of rare mutations not detected by this test

    All markers should be interpreted as a group and not individually

    FLT3 Mutation Detection by PCR 2005400
    Method: Qualitative Polymerase Chain Reaction/Capillary Electrophoresis

    Limitations

    Negative test result does not exclude presence of mutations in transcripts below the detection limit, presence of rare mutations not detected by this test

    All markers should be interpreted as a group and not individually

    FLT3 Signal Ratio Mutation Detection by PCR 2011806
    Method: Qualitative Polymerase Chain Reaction/Capillary Electrophoresis

    Limitations

    Negative test result does not exclude presence of mutations in transcripts below the detection limit, presence of rare mutations not detected by this test

    All markers should be interpreted as a group and not individually

    IDH1 and IDH2 Mutation Analysis, exon 4 2006444
    Method: Polymerase Chain Reaction/Sequencing

    Limitations

    Negative test result does not exclude mutations below the limit of detection, presence of mutations other than those detected by the test

    This marker should be interpreted within the group of CN-AML prognostic markers

    WT1 Mutation Detection by Sequencing 2005766
    Method: Polymerase Chain Reaction/Sequencing

    KIT Mutations in AML by Fragment Analysis and Sequencing 2002437
    Method: Polymerase Chain Reaction/Fragment Analysis/Sequencing

    Limitations

    Not intended to detect minimal residual disease

    Mutations outside of exons 8 and 17 are not detected

    Mutations below analytical sensitivity will not be detected

    Eosinophilia Panel by FISH 2002378
    Method: Fluorescence in situ Hybridization

    Limitations

    Detects only rearrangements targeted by the probes

    PDGFRB gene on 5q33 and FGFR1 gene on 8p11 have multiple translocation partners; translocation partners are not identified by this test

    CD19 by Immunohistochemistry 2005114
    Method: Immunohistochemistry

    CD34, QBEnd/10 by Immunohistochemistry 2003556
    Method: Immunohistochemistry

    CD68, KP1 by Immunohistochemistry 2003598
    Method: Immunohistochemistry

    CD79A by Immunohistochemistry 2003800
    Method: Immunohistochemistry

    CD117 (c-Kit) by Immunohistochemistry 2003806
    Method: Immunohistochemistry

    Myeloperoxidase Stain 0049030
    Method: Cytochemical Stain

    Pax-5 by Immunohistochemistry 2004082
    Method: Immunohistochemistry

    Lysozyme (Muramidase) by Immunohistochemistry 2003990
    Method: Immunohistochemistry

    Additional Tests Available

    PML-RARA Translocation, t(15;17) by RT-PCR, Quantitative 2002871
    Method: Reverse Transcription Polymerase Chain Reaction

    Comments

    May be used for genetic confirmation of APL; however, FISH is more rapid and is the recommended test

    Access therapeutic response, predict relapse risk, and monitor for MRD postconsolidation therapy

    Primer sets are designed to detect all 3 gene fusion patterns: type A (short, S-form, bcr-3), type B (long, L-form, bcr-1) and type B variant (variable, V-form, bcr-2)

    Bone marrow samples preferred for maximum sensitivity

    Poor RNA yield will lead to false negatives (more common with peripheral blood)

    Translocations involving other genes or gene partners will not be detected

    Clinical sensitivity – >97%

    Clinical specificity – essentially diagnostic for APL in the correct clinical context

    Analytical sensitivity – 1 in 104

    Analytical specificity – 85%

    Chromosome Analysis, Bone Marrow 2002292
    Method: Giemsa Band

    Comments

    Diagnosis, prognosis, and monitoring of AML

    Chromosome Analysis, Leukemic Blood 2002290
    Method: Giemsa Band

    Comments

    Detect chromosome abnormalities in peripheral blood consistent with diagnosis of AML

    Prognosis in AML

    Number of dividing cells in the peripheral blood may be insufficient to allow for full analysis of metaphase cells; bone marrow aspirate may be more informative

    CBC with Platelet Count 0040002
    Method: Automated Cell Count

    Comments

    Screen for blasts and cytopenias

    Prothrombin Time 0030215
    Method: Electromagnetic Mechanical Clot Detection

    Comments

    Screen for DIC related to acute promyelocytic leukemia

    Partial Thromboplastin Time 0030235
    Method: Electromagnetic Mechanical Clot Detection

    Comments

    Screen for DIC related to acute promyelocytic leukemia

    Fibrinogen 0030130
    Method: Electromagnetic Mechanical Clot Detection

    Comments

    Screen for DIC related to acute promyelocytic leukemia

    D-Dimer 0030057
    Method: Immunoturbidimetry

    Comments

    Screen for DIC related to acute promyelocytic leukemia

    Myeloperoxidase (MPO) by Immunohistochemistry 2004014
    Method: Immunohistochemistry

    Comments

    Aid in the histologic diagnosis of AML

    Stained and returned to client pathologist for interpretation; consultation available if needed

    IDH1 R132H Point Mutation with Interpretation by Immunohistochemistry 2007357
    Method: Immunohistochemistry

    Comments

    Sequencing is preferred

    Guidelines

    Döhner H, Estey E, Amadori S, Appelbaum F, Büchner T, Burnett A, Dombret H, Fenaux P, Grimwade D, Larson R, Lo-Coco F, Naoe T, Niederwieser D, Ossenkoppele G, Sanz M, Sierra J, Tallman M, Lowenberg B, Bloomfield C, European LeukemiaNet. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood. 2010; 115(3): 453-74. PubMed

    NCCN Clinical Practice Guidelines in Oncology, Acute Myeloid Leukemia. National Comprehensive Cancer Network. Fort Washington, PA [Accessed: Jun 2015]

    General References

    Döhner H, Gaidzik V. Impact of genetic features on treatment decisions in AML. Hematology Am Soc Hematol Educ Program. 2011; 2011: 36-42. PubMed

    Jabbour E, Estey E, Kantarjian H. Adult acute myeloid leukemia. Mayo Clin Proc. 2006; 81(2): 247-60. PubMed

    Lin T, Smith D. Prognostically important molecular markers in cytogenetically normal acute myeloid leukemia. Am J Med Sci. 2011; 341(5): 404-8. PubMed

    Morrissette J, Bagg A. Acute myeloid leukemia: conventional cytogenetics, FISH, and moleculocentric methodologies. Clin Lab Med. 2011; 31(4): 659-86, x. PubMed

    Sanz M, Grimwade D, Tallman M, Lowenberg B, Fenaux P, Estey E, Naoe T, Lengfelder E, Büchner T, Döhner H, Burnett A, Lo-Coco F. Management of acute promyelocytic leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet. Blood. 2009; 113(9): 1875-91. PubMed

    Schoch C. M3/M3v acute myeloid leukemia (AML M3/M3v); Acute promyelocytic leukemia (APL). Atlas of Genetics and Cytogenetics in Oncology and Haematology. Poitiers, France [Accessed: Nov 2015]

    Sun T. Clinical Application: Case 6.. In Pine J, McGough J, et al, eds. Flow Cytometry and Immunohistochemistry for Hematologic Neoplasms, 2nd ed.. Philadelphia, PA: Lippincott Williams & Wilkins, 2011.

    Swerdlow S, Campo E, Harris N, Jaffe E, Pileri S, Harald S, Thiele J, Vardiman J. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th ed. Lyon, France: International Agency for Research on Cancer, 2008.

    Watt C, Bagg A. Molecular diagnosis of acute myeloid leukemia. Expert Rev Mol Diagn. 2010; 10(8): 993-1012. PubMed

    Zhou Y, Jorgensen J, Wang S, Ravandi F, Cortes J, Kantarjian H, Medeiros J, Konoplev S. Usefulness of CD11a and CD18 in flow cytometric immunophenotypic analysis for diagnosis of acute promyelocytic leukemia. Am J Clin Pathol. 2012; 138(5): 744-50. PubMed

    References from the ARUP Institute for Clinical and Experimental Pathology®

    Chen Z, Pasquini M, Hong B, DeHart S, Heikens M, Tsai S. The human Penumbra gene is mapped to a region on chromosome 7 frequently deleted in myeloid malignancies. Cancer Genet Cytogenet. 2005; 162(2): 95-8. PubMed

    Koshy J, Qian Y, Bhagwath G, Willis M, Kelley T, Papenhausen P. Microarray, gene sequencing, and reverse transcriptase-polymerase chain reaction analyses of a cryptic PML-RARA translocation. Cancer Genet. 2012; 205(10): 537-40. PubMed

    Prchal J. Molecular basis of polycythemic disorders due to aberrant hypoxia sensing and its relevance to acute leukemia. Best Pract Res Clin Haematol. 2012; 25(4): 493-7. PubMed

    Ridges S, Heaton W, Joshi D, Choi H, Eiring A, Batchelor L, Choudhry P, Manos E, Sofla H, Sanati A, Welborn S, Agarwal A, Spangrude G, Miles R, Cox J, Frazer K, Deininger M, Balan K, Sigman M, Müschen M, Perova T, Johnson R, Montpellier B, Guidos C, Jones D, Trede N. Zebrafish screen identifies novel compound with selective toxicity against leukemia. Blood. 2012; 119(24): 5621-31. PubMed

    Spencer D, Abel H, Lockwood C, Payton J, Szankasi P, Kelley T, Kulkarni S, Pfeifer J, Duncavage E. Detection of FLT3 internal tandem duplication in targeted, short-read-length, next-generation sequencing data. J Mol Diagn. 2013; 15(1): 81-93. PubMed

    Talpaz M, Paquette R, Afrin L, Hamburg S, Prchal J, Jamieson K, Terebelo H, Ortega G, Lyons R, Tiu R, Winton E, Natrajan K, Odenike O, Claxton D, Peng W, O'Neill P, Erickson-Viitanen S, Leopold L, Sandor V, Levy R, Kantarjian H, Verstovsek S. Interim analysis of safety and efficacy of ruxolitinib in patients with myelofibrosis and low platelet counts. J Hematol Oncol. 2013; 6(1): 81. PubMed

    Tomasic N, Piterkova L, Huff C, Bilic E, Yoon D, Miasnikova G, Sergueeva A, Niu X, Nekhai S, Gordeuk V, Prchal J. The phenotype of polycythemia due to Croatian homozygous VHL (571C>G:H191D) mutation is different from that of Chuvash polycythemia (VHL 598C>T:R200W). Haematologica. 2013; 98(4): 560-7. PubMed

    Yang D, Greenwood J, Hartung L, Hill S, Perkins S, Bahler D. Flow cytometric analysis of different CD14 epitopes can help identify immature monocytic populations. Am J Clin Pathol. 2005; 124(6): 930-6. PubMed

    Medical Reviewers

    Last Update: January 2016