Acute Myeloid Leukemia - AML

Key Points

Cytogenetically-Normal AML (CN-AML) Prognostic Markers 

Cytogenetic analysis is the initial test for risk stratification in AML. Although 50% of AML cases are cytogenetically normal (CN-AML) and considered to be intermediate risk, single gene mutations exist that provide subtype stratification within this risk group. Use of these new 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 samples and used for additional testing after determination of initial cytogenetic risk classification.

For molecular markers in CN-AML, see table below.

MOLECULAR MARKERS IN CN-AML

Marker

Biology

Prognosis

Well-Characterized Markers

NPM1 mutation

ARUP available tests:

Whole blood or bone marrow

Paraffin  

Nucleolar phosphoprotein that shuttles between nucleus and cytoplasmNPM1 mutations associated with favorable outcome

CEPBA mutations

ARUP available tests:

Whole blood or bone marrow

Transcription factor involved in myeloid differentiation

Double CEPBA mutations associated with favorable outcome

Single CEPBA mutations have unclear clinical significance

FLT3 mutations

ARUP available tests:

Whole blood or bone marrow  

Tyrosine kinase receptor expressed by hematopoietic progenitor cells that regulates cell survival and maturation

FLT3 internal tandem mutations (ITD) associated with unfavorable outcome

FLT3 D385 (tyrosine kinase domain mutations) associated with unfavorable outcome

Investigational Markers

IDH1 mutation, IDH2 mutation

Enzyme involved in citric acid cycle

Mutations cause neomorphic activity that leads to production of the onco metabolite 2-hydroxyglutarate

IDH1 SNP rs11554137 associated with unfavorable outcome, particularly if associated with FLT3 or NMP1 wild type mutations

WT1 mutation

ARUP available tests:

Whole blood or bone marrow  

Transcription factor expressed in embryonic kidney cells and hematopoietic cells that has both tumor suppressor and oncogenic functions

Mutations of exon 7 or 9 associated with unfavorable outcome

G-allele of SNP rs16754 in exon 7 associated with improved outcome in patients with FLT3 and wild-type NPM1

DNMT3A mutation

Encodes for methyltransferases

Mutations cause reduced enzymatic activity with aberrant DNA methylation

DNMT3A mutation associated with unfavorable outcome

BAALC overexpression

Expressed in CD34 positive hemopoietic progenitors and neural tissue; functions in cytoskeletonOverexpression associated with unfavorable outcome

MLL partial tandem duplication (PTD) mutations, overexpression

Involved in chromatin modificationMLL PTD associated with less favorable outcome

BAALC – brain and acute leukemia, cytoplasmic; CEPBA – CAAT/enhancer binding protein (C/EBP) α; DNMT3A – DNA (cytosine-5-)-methyltransferase 3 alpha; FLT3 – FMS-related tyrosine kinase 3; IDH1 – isocitrate dehydrogenase 1 (NADP+), soluble; IDH2 – isocitrate dehydrogenase 2 (NADP+), mitochondrial; MLL – myeloid/lymphoid or mixed-lineage leukemia; NPM1 – nucleophosmin (nucleolar phosphoprotein B23, numatrin); WT1 – Wilms tumor 1

Dohner, H, 2011; Lin et al, 2011; Watt et al, 2010.

Diagnosis

Indications for Testing

  • Findings of 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 present (typically promyelocytic leukemias)
    • D dimer if DIC is suspected

Histology

  • Combination of methods used include cytomorphology, cytochemistry, and flow cytometric immunophenotyping
    • Key distinction is between acute lymphoblastic leukemia (ALL) and AML
      • AML will demonstrate mostly myeloid markers without expressing 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 with fewer blasts
    • Cytogenetics – provides genetic classification of leukemia
      • Usually assessed using PCR, SNP microarray, and FISH testing
      • Classification
        • AML-M2 – t(8;21)
        • AML-M3 (promyelocytic) – t(15;17)
        • AML-M4 – t(16;16) or inv(16)
        • AML-M5a/5b – 11q23 (MLL) rearrangement
        • AML-M6 – no consistent cytogenetic patterns; frequent 5 and 7 deletions due to high incidence of therapy-related leukemias
        • AML with t(6;9)
        • AML with inv(3) or t(3;3)
        • AML-M7 (megakaryoblastic) with t(1;22)
        • Therapy-related AML (similar cytogenetic abnormalities can also be observed in primary AML) 
          • Alkylating agent/radiation therapy – -7 or del(7q), -5 or del(5q) and/or complex and nonspecific chromosome abnormalities
          • Topoisomerase II inhibitor-related therapy – 11q23 (MLL) rearrangement, inv(16), t(8;21), t(8;16), and t(6;9)
      • SNP microarray cannot test for t(15;17) (PML/RARα), t(8;21) (RUNX1T1/RUNX1) and balanced rearrangement of MLL or CBFβ gene
    • 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+
    • Immunohistochemistry
      • Most useful stains include CD19, CD34, CD68, CD79a, CD117, lysozyme, myeloperoxidase, and Pax-5

Prognosis

  • Dependent on karyotyping, type of AML, molecular abnormalities
  • Karyotyping
    • Favorable 
      • t(8,21) – AML1-ETO
      • inv(16) – CBFβ-MYH11
      • t(15;17) – PML-RARα
    • Intermediate
      • Normal karyotype
      • Diploid
      • +8
      • del(12p)
      • +6
      • -Y
      • t(9;11) – MLL-AF4, 11q23
    • Unfavorable
      • 11q23 – non t(9;11)
      • Philadelphia+ (Ph+) t(9;22)
      • -5/del(5q)
      • -7/del(7q)
      • Abnormal 3q, 9q, 11q, 20q, 21q, 17p
      • t(6;9) – DEK-CAN
      • inv(3) or t(3;3) – EVI1
      • Complex karyotypes – ≥3 clonal abnormalities
  • Type of AML
    • Favorable
      • Promyelocytic
      • Megakaryocytic in Down syndrome
  •  Molecular marker testing for cytogenetically normal AML (CN-AML) – see Key Points section
    • Favorable 
      • NPM1
      • Double CEBPα
      • WT1 G-allele of SNP rs16754 in exon 7 patients with FLT3 and wild-type NPM1
    • Unfavorable
      • Internal tandem FLT3 
      • IDH1, IDH2
      • WT1
      • MLL partial tandem duplication
      • BAALC overexpression
      • DNMT3A

Differential Diagnosis

  • ALL
  • Leukemoid reaction (seen with infection)
  • MDS
  • MPN

Monitoring

  • Bone marrow biopsy with PCR, FISH 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%; lower levels will likely appear normal
      • Depending on clinical setting, flow cytometry or PCR test may be informative
    • Immunophenotyping
    • Specific monitoring – RT-PCR quantitative testing for PML-RARα t(15;17)
      • Monitor acute promyelocytic leukemia (APML)
  • Response criteria for AML

    Response Criteria for AML*

    Morphologic leukemia-free state

    • Bone marrow <5% blasts in aspirate with spicules
    • No blasts with Auer rods or persistence of extramedullary disease
    • Question of residual leukemia
      • Repeat bone marrow aspirate/biopsy in one week
      • Perform bone marrow biopsy if no spicules found in aspirate sample

    Complete remission (CR)

    • Morphologic CR – patient independent of transfusions
      • Absolute neutrophil count >1,000/mcL
      • Platelets ≥100,000/mcL
      • No evidence of residual extramedullary disease
    • Cytogenetic CR – cytogenetics normal (in those with previously abnormal cytogenetics)
    • Molecular CR – molecular studies negative (for promyelocytic and Ph+ only)

    Partial remission (useful only in Phase I trials and not as a goal for standard therapy)

    • Decrease of ≥50% in percentage of blasts in bone marrow aspirate (5-25%)
    • Normalization of blood counts, as noted in morphologic CR

    No remission

    • Patient fails to achieve complete response – considered treatment failure

    Relapse following complete response

    • Defined as reappearance of leukemic blasts in peripheral blood or finding of >5% blasts in bone marrow not attributable to another cause (eg, bone marrow regeneration after consolidation therapy) or extramedullary relapse

    *Adapted from revised recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia, 2003

Clinical Background

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 – 2.7/100,000
  • Age – peaks in the 50s-60s
  • Sex – M>F (minimal)

Current Classification

  • Based on blast cytogenetic features, differentiation and morphology
  • Genetic studies are important in classification and prognosis
  • WHO classification of acute myeloid leukemia – based on genetics and morphology
    WHO Classification of Acute Myeloid Leukemia*

    Acute myeloid leukemia with recurrent genetic abnormalities

    • Acute myeloid leukemia with t(8;21)(q22;q22), (AML1-ETO)
    • Acute myeloid leukemia with abnormal bone marrow eosinophils and inv(16)(p13q22) or t(16;16)(p13;q22), (CBFβ-MYH11)
    • Acute promyelocytic leukemia with t(15;17)(q22;q12), (PML-RARα) and variants (M3)
    • Acute myeloid leukemia with 11q23 (MLL) abnormalities

    Acute myeloid leukemia with multilineage dysplasia

    Acute myeloid leukemia and myelodysplastic syndromes, therapy related

    • Alkylating agent/radiation-related type
    • Topoisomerase II inhibitor-related type (some may be lymphoid)
    • Others

    Acute myeloid leukemia, not otherwise categorized

    • Acute myeloid leukemia, minimally differentiated (M0)
    • Acute myeloid leukemia without maturation (M1)
    • Acute myeloid leukemia with maturation (M2)
    • Acute myelomonocytic leukemia (M4)
    • Acute monoblastic (M5a)/acute monocytic leukemia (M5b)
    • Acute erythroid leukemia (erythroid/myeloid and pure erythroleukemia) (M6)
    • Acute megakaryoblastic leukemia (M7)
    • Acute basophilic leukemia
    • Acute panmyelosis with myelofibrosis
    • Myeloid sarcoma
    *Defined as ≥20% blasts in blood or bone marrow; however, clonal, recurring cytogenetic abnormalities should be considered AML regardless of blast percentage. Ongoing clinical trials may continue to use French-American-British (FAB) criteria of ≥30% blasts until completion of trial. FAB classification identified as M0 through M7.

Risk Factors

  • Myeloproliferative neoplasm (MPN) or myelodysplastic syndrome (MDS) – chronic myelogenous leukemia, polycythemia vera, essential thrombocythemia, idiopathic myelofibrosis
  • Environmental risk factors – radiation, benzene, paint, pesticides
  • Genetic – Down syndrome, Fanconi anemia, Bloom syndrome, Wiskott-Aldrich syndrome, sibling with AML
  • Drugs – cytotoxic chemotherapy (eg, alkylating agents, topoisomerase II inhibitors)

Pathophysiology

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

Clinical Presentation

  • Nonspecific symptoms such as weakness, fever, bruising, weight loss
  • 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
Test Name and Number Recommended Use Limitations Follow Up
Chromosome Analysis, Leukemic Blood 2002290
Method: Giemsa Band

Detect chromosome abnormalities in peripheral blood consistent with diagnosis of AML

Prognosticate 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

  
Chromosome Analysis, Bone Marrow 2002292
Method: Giemsa Band

Detect chromosome abnormalities in peripheral blood consistent with diagnosis of AML

Prognosticate in AML

  

Repeat testing as clinically indicated to monitor disease progression
Leukemia/Lymphoma Phenotyping (Comprehensive - Whole Blood) 0096299
Method: Flow Cytometry

Detect chromosome abnormalities in peripheral blood consistent with diagnosis of AML

Prognosticate in AML

    
Leukemia/Lymphoma Phenotyping (Comprehensive - Bone Marrow) 0095244
Method: Flow Cytometry

Detect chromosome abnormalities in peripheral blood consistent with diagnosis of AML

Prognosticate in AML

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

Detect chromosome abnormalities in peripheral blood consistent with diagnosis of AML

Prognosticate in AML

    
Chromosome FISH, Interphase 2002298
Method: Fluorescence in situ Hybridization

Use to detect diagnostic and prognostic abnormalities

Monitor disease and identify specific abnormalities suspected clinically

Specific FISH probes must be requested; for this indication include t(15;17), t(8;21), inv(16), 11q23 rearrangements, monosomy 7 or 7q deletion, 5q deletion, +8, and 20q deletion

Indicate names of probes needed for testing 

ARUP Oncology FISH Probes menu

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 MDS and MPN and therefore are not by themselves sufficient for diagnosis but rather consistent with the suspected diagnosis

Repeat testing as clinically indicated to monitor disease progression
Acute Myelogenous Leukemia Panel by FISH 2002384
Method: Fluorescence in situ Hybridization

Identify prognostically important translocations in newly diagnosed AML

Probes included are RUNX1T1-RUNX1, MLL, and CBFβ

PML-RARα performed and reported separately

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

Provides prognostic information for patients with AML arising in the setting of MDS or patients with therapy-related MDS/AML

Test also aids in monitoring response to therapy or progression of disease

EGR1 (5q del), D7S486 (7q del/-7), MLL probes are included

Chromosome alterations outside regions complementary to these probes are not detected

  
PML/RARa FISH 2002363
Method: Fluorescence in situ Hybridization

Use to diagnose promyelocytic leukemia by detection of t(15;17)

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

Identify PML-RARα fusion transcript in diagnosis of promyelocytic leukemia

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)

Limit of detection is 1/1,000 cells; does not detect minimal residual disease

  
CBFB-MYH1, inv(16) by RT-PCR 0092209
Method: Reverse Transcription Polymerase Chain Reaction

Diagnose AML with abnormal bone marrow eosinophils and inv(16)

Test not intended to detect minimal residual disease

Should not be used alone for diagnosis of malignancy

Negative result does not preclude presence of CBFB-MYH11 fusion transcripts below limit of detection or presence of type other than A, D or E

  
RUNX1-RUNX1T1 (AML1-ETO) Translocation, t(8;21) by RT-PCR 0050444
Method: Reverse Transcription Polymerase Chain Reaction

Identify type of AML

Monitor minimal residual disease

Negative result does not exclude the presence of the t(8;21)

Test result must always be interpreted in the context of morphologic and other relevant data and should not be used alone for a diagnosis of malignancy

  
CEBPA Mutation Detection 2004247
Method: Polymerase Chain Reaction/Sequencing

Prognosis in CN-AML

 Negative result does not preclude the presence of CEBPA mutations in rare AML cells below the detection limit of this test  
NPM1 Mutation by PCR and Fragment Analysis, Fluid 0040174
Method: Polymerase Chain Reaction/Fragment Analysis

Prognosis in CN-AML

Should not be used alone for diagnosis of malignancy

Negative result does not preclude the presence of NPM1 mutations in rare AML cells below the detection limit of this test

  
NPM1 Mutation by PCR and Fragment Analysis, Paraffin 0040179
Method: Varies

Prognosis in CN-AML

Should not be used alone for diagnosis of malignancy

Negative result does not preclude the presence of NPM1 mutations in rare AML cells below the detection limit of this test

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

Prognosis in CN-AML

Negative result does not exclude presence of FLT3 gene mutation

Assay is a qualitative test and should not be used to detect minimal residual disease

Should not be used alone for diagnosis of malignancy

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

Prognosis cytogenetically abnormal AML with t(8;21) or inv(16)

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

Prognosis in CN-AML

    
CD19 by Immunohistochemistry 2005114
Method: Immunohistochemistry

Aid in histologic diagnosis of B-cell leukemia/lymphoma

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

    
CD34, QBEnd/10 by Immunohistochemistry 2003556
Method: Immunohistochemistry

Aid in the histologic diagnosis of AML

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

    
CD68, KP1 by Immunohistochemistry 2003598
Method: Immunohistochemistry

Aid in the histologic diagnosis of AML

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

    
CD79A by Immunohistochemistry 2003800
Method: Immunohistochemistry

Aid in the histologic diagnosis of AML

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

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

Aid in the histologic diagnosis of AML

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

    
Myeloperoxidase (MPO) by Immunohistochemistry 2004014
Method: Immunohistochemistry

Aid in the histologic diagnosis of AML

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

    
Pax-5 by Immunohistochemistry 2004082
Method: Immunohistochemistry

Aid in the histologic diagnosis of AML

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

    
Lysozyme (Muramidase) by Immunohistochemistry 2003990
Method: Immunohistochemistry

Aid in the histologic diagnosis of AML

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

    
Eosinophilia Panel by FISH 2002378
Method: Fluorescence in situ Hybridization

Diagnose/provide prognostic information in patients with AML with eosinophilia

Probes include PDGFRα, PDGFRβ, FGFR1 and CBFB

    
Additional Tests Available
 
 
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
CBC with Platelet Count 0040002
Method: Automated Cell Count

Screen for blasts and cytopenias
Prothrombin Time 0030215
Method: Electromagnetic Mechanical Clot Detection

Screen for DIC related to acute promyelocytic leukemia
Partial Thromboplastin Time 0030235
Method: Electromagnetic Mechanical Clot Detection

Screen for DIC related to acute promyelocytic leukemia
Fibrinogen 0030130
Method: Electromagnetic Mechanical Clot Detection

Screen for DIC related to acute promyelocytic leukemia
D-Dimer 0030057
Method: Immunoturbidimetry

Screen for DIC related to acute promyelocytic leukemia
Esterase Stain, Nonspecific 0049050
Method: Cytochemical Stain

Determine monocytic lineage in AML per WHO classification

Less sensitive and specific than flow cytometry immunophenotype
Esterase Stain, Specific 0049055
Method: Cytochemical Stain

Determine myeloid lineage in blasts of acute leukemias

Less sensitive and specific than flow cytometry immunophenotype
Sudan Black B Stain 0049040
Method: Stain

Determine myeloid lineage

Less sensitive and specific than flow cytometry immunophenotype
Myeloperoxidase Stain 0049030
Method: Cytochemical Stain

Determine myeloid lineage in AML per WHO classification

Less sensitive and specific than flow cytometry immunophenotype
Periodic Acid Schiff Stain 0049045
Method: Stain

Use to differentiate ALL from AML

Less sensitive and specific than flow cytometry immunophenotype