Acute Myeloid Leukemia

Background

Acute myeloid leukemia (AML) is a malignant neoplasm (cancer) 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 AML is based on genetics and morphology

WHO Classification of Acute Myeloid Leukemia

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 AML following myelodysplastic syndrome (MDS) or (MDS)/myeloproliferative neoplasm (MPN) AML without antecedent MDS or MDS/MPN but with dysplasia in at least 50% of cells in 2 or more myeloid lineages 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.

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

AML following myelodysplastic syndrome (MDS) or (MDS)/myeloproliferative neoplasm (MPN)
AML without antecedent MDS or MDS/MPN but with dysplasia in at least 50% of cells in 2 or more myeloid lineages

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

Transformation of 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 (ie, 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
- Tumor lysis syndrome
  - Associated with white blood cell (WBC) count >100x10⁹/L
  - Hyperuricemia, renal failure, acidosis, hypocalcemia and hyperphosphatemia
  - May be spontaneous or treatment-mediated cell destruction
  - Oncologic emergency
- Disseminated intravascular coagulation (DIC) – acute promyelocytic leukemia with t(15;17), AML-M3
- Leukostasis associated with WBC >100x10⁹/L
  - Dyspnea, chest pain and headache
  - Oncologic emergency

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
- D-dimer if DIC is suspected (typically occurs in promyelocytic leukemia)

Histology

Combination of methods is used, including cytomorphology, cytochemistry and flow cytometric immunophenotyping
- 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
    - AML-M2 – t(8;21); follow up with PCR and FISH
    - AML-M3 (promyelocytic) – t(15;17); follow up with PCR and FISH
    - AML-M4 – t(16;16) or inv(16); follow up with PCR and FISH
    - AML-M5a/5b – 11q23 (MLL) rearrangement; follow up with PCR and FISH
    - 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)
- Flow cytometry – establish myeloid or monocytic lineage
- 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
- Immunohistochemistry – not as practical as flow cytometry
  - Most useful stains include CD19, CD34, CD68, CD79a, CD117, lysozyme, myeloperoxidase and Pax-5

Prognosis

Karyotyping
- Favorable
  - t(8,21) – AML1-ETO
  - inv(16) – CBFβ-MYH11
  - t(15;17) – PML-RARA
- 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) – EVI-1
  - Complex karyotypes – ≥3 clonal abnormalities
Mutations
- Favorable
  - NPM1 – if concomitant FLT3-ITD mutation present, then intermediate prognosis
  - CEBPA – if present in patients with either intermediate risk cytogenetics or normal karyotype
- Unfavorable
  - KIT – in patients with AML and t(8;21) or inv(16)/t(16;16) abnormalities
  - FLT3-ITD (with normal karyotype) – if specific mutations (such as internal tandem repeats) are present
  - MLL-PTD – if present
  - BAALC – for high-level expression
  - MN1 – for high-level expression

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)
- RT-PCR quantitative testing for PML-RARA t(15;17) – monitor acute promyelocytic leukemia (APML)
- 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

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

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

Tests

Tests generally appear in the order most useful for common clinical situations

Test name: CBC with Platelet Count

ARUP #: 0040002

Methodology: Automated Cell Count

Use:

Screen for blasts and cytopenias

Test name: Prothrombin Time

ARUP #: 0030215

Methodology: Electromagnetic Mechanical Clot Detection

Use:

Screen for promyelocytic leukemia related to DIC

Test name: Partial Thromboplastin Time

ARUP #: 0030235

Methodology: Electromagnetic Mechanical Clot Detection

Use:

Screen for promyelocytic leukemia related to DIC

Test name: Fibrinogen

ARUP #: 0030130

Methodology: Electromagnetic Mechanical Clot Detection

Use:

Screen for promyelocytic leukemia related to DIC

Test name: D-Dimer

ARUP #: 0030057

Methodology: Immunoturbidimetric

Use:

Screen for promyelocytic leukemia related to DIC

Test name: Chromosome Analysis, Leukemic Blood

ARUP #: 2002290

Methodology: Giemsa-Band Analysis

Use:

Detect chromosome abnormalities in peripheral blood consistent with diagnosis of AML; some abnormalities also have classification and prognostic significance

Limitations:

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

Test name: Leukemia/Lymphoma Phenotyping (Comprehensive - Whole Blood)

ARUP #: 0096299

Methodology: Flow Cytometry

Use:

Detect chromosome abnormalities in peripheral blood consistent with diagnosis of AML; some abnormalities also have classification and prognostic significance

Test name: Leukemia/Lymphoma Phenotyping (Comprehensive - Bone Marrow)

ARUP #: 0095244

Methodology: Flow Cytometry

Use:

Detect chromosome abnormalities in bone marrow aspirate consistent with diagnosis of AML; some abnormalities also have classification and prognostic significance

Test name: Chromosome Analysis, Bone Marrow

ARUP #: 2002292

Methodology: Giemsa-Band Analysis

Use:

Detect chromosome abnormalities in bone marrow aspirate consistent with diagnosis of AML some abnormalities also have classification and prognostic significance

Follow-up:

Repeat testing as clinically indicated to monitor disease progression

Test name: Chromosome FISH, Interphase

ARUP #: 2002298

Methodology: Fluorescence in situ Hybridization

Use:

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

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 MDS and 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

Test name: Acute Myelogenous Leukemia Panel by FISH

ARUP #: 2002384

Methodology: Fluorescence in situ Hybridization

Use:

Identify prognostically important translocations in newly diagnosed AML

Probes included are RUNX1T1-RUNX1, MLL, and CBFB

PML-RARalpha performed and reported separately

Test name: Acute Myelogenous Leukemia (AML) with Myelodysplastic Syndrome (MDS), or Therapy-Related AML, by FISH

ARUP #: 2002653

Methodology: Fluorescence in situ Hybridization

Use:

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

Limitations:

Chromosome alterations outside regions complementary to these probes are not detected

Test name: PML/RARa FISH

ARUP #: 2002363

Methodology: Fluorescence in situ Hybridization

Use:

Diagnose promyelocytic leukemia by detection of t(15;17)

Test name: PML-RARA Translocation, t(15;17) by RT-PCR, Quantitative

ARUP #: 2002871

Methodology: Reverse Transcription Polymerase Chain Reaction

Use:

Identify PML-RARA fusion transcript

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)

Limitations:

Limit of detection is 1/1000 cells; test does not detect minimal residual disease

Test name: CBFB-MYH1, inv(16) by RT-PCR

ARUP #: 0092209

Methodology: Reverse Transcription Polymerase Chain Reaction

Use:

Detect presence of CBFB-MYH11 fusion transcripts in patients with AML-M4Eo or an indication of inv(16)

Limitations:

Test not intended to detect minimal residual disease

Results must be interpreted in context of morphologic and other relevant data

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

Test name: RUNX1-RUNX1T1 (AML1-ETO) Translocation, t(8;21) by RT-PCR

ARUP #: 0050444

Methodology: Reverse Transcription Polymerase Chain Reaction

Use:

Monitor persistent disease and when a diagnosis is suspected but standard cytogenetics are not available or do not show the abnormality

Identify AML type FAB-M2

Monitor minimal residual disease

Limitations:

Negative result does not entirely 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

Test name: CEBPA Mutation Detection

ARUP #: 2004247

Methodology: Polymerase Chain Reaction/Sequencing

Use:

May be useful in prognosis

Limitations:

Results must be interpreted in context of morphologic and other relevant data

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

Test name: NPM1 Mutation by PCR and Fragment Analysis, Fluid

ARUP #: 0040174

Methodology: Polymerase Chain Reaction/ Fragment Analysis

Use:

May be useful in prognosis

Limitations:

Results must be interpreted in context of morphologic and other relevant data

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

Test name: NPM1 Mutation by PCR and Fragment Analysis, Paraffin

ARUP #: 0040179

Methodology: Polymerase Chain Reaction/ Fragment Analysis

Use:

Detect mutations in the NPM1 gene associated with AML prognosis

Limitations:

Results must be interpreted in context of morphologic and other relevant data

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

Test name: FLT3 Mutation Detection by PCR

ARUP #: 2005400

Methodology: Qualitative Polymerase Chain Reaction/Capillary Electrophoresis

Use:

Detect mutations in FLT3 gene in patient with AML

Limitations:

Negative result does not entirely exclude presence of FLT3 gene mutation

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

Must be interpreted in context of clinicopathologic and other relevant data

Should not be used alone for diagnosis of malignancy

Test name: KIT Mutations in AML by Fragment Analysis and Sequencing

ARUP #: 2002437

Methodology: Polymerase Chain Reaction/Fragment Analysis/Sequencing

Use:

Detect mutations in the KIT gene, which are associated with a worse prognosis in patients with AML with t(8;21) or inv(16)

Test name: BCL-6 by Immunohistochemistry

ARUP #: 2003457

Methodology: Immunohistochemistry

Use:

Aid in the histologic diagnosis of AML