Acute Myeloid Leukemia - AML

Key Points

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 hemorrhages. Rapid diagnosis is imperative to reduce 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 the PML-RARA fusion is mandatory in all cases.

Clinical Findings

Bleeding, bruising, pallor (due to cytopenias that may be profound) Combination of symptoms is common Life threatening coagulopathies (DIC) may occur Bleeding secondary to DIC – unique to APL

Morphology

Presence of atypical promyelocytes in peripheral blood and bone marrow Peripheral blood Circulating blasts may be few in number Thrombocytopenia – often severe 2 morphological variants Microgranular variant – more likely associated with leukocytosis Hypergranular variant – more likely associated with leukopenia Bone marrow  Blasts (atypical promyelocytes) are myeloperoxidase (strong, uniform) positive Significant infiltration with atypical promyelocytes Hypergranular variant – often multiple Auer rods in a single cell Microgranular variant – bilobed, reniform, or monocyte-like nuclei

Immunophenotype (flow cytometry)

Atypical promyelocytes of APL typically express CD33 and CD117 without CD34 or HLA-DR and have slightly higher side scatter characteristics than normal myeloblasts Microgranular variant may also show aberrant expression of CD2 Immunophenotypic features Myelomonocytic antigens Positive – CD13, CD33 Negative – CD14 Granulocyte differentiation markers Negative or weakly expressed – CD15,  CD65 Positive – CD64 Leukocyte integrin markers Low or absence of – CD11a, CD11b, CD11c Positive – CD33, CD64, CD117

Molecular Biology

APL with t(15;17)(q24.1;q21.1) – PML-RARA Reciprocal translocation between the long arms of chromosomes 15 and 17 with the creation of a fusion gene (PML-RARA) which links the retinoic acid receptor alpha (RARA) gene on chromosome 17 with the PML gene on chromosome 15   Chromosomal banding Variable breakpoints reported – t(15;17)(q24;q21); t(15;17)(q22;q12) Rarely t(5;17) or t(11;17), indicating RARA with alternative partners

Laboratory Tests for Genetic Confirmation of the PML-RARA Fusion

Laboratory Tests for Genetic Confirmation of the PML-RARA Fusion
Fluorescent in situ hybridization Highly efficient for confirming APL ARUP  available tests PML-RARα FISH 2002363 Acute Myelogenous Leukemia Panel by FISH 2002384	Test Characteristics Probes designed to check fusion of PML and RARA Bone marrow preferred; however,  peripheral blood can be used when hyperleukocytosis is present Advantages Can be performed within 24 hrs High sensitivity, specificity Less expensive than and time-consuming when compared to karyotyping Can raise suspicion of rare subtypes of APL (eg, t(11;17), t(5;17)) Disadvantages May take up to 2 days for results False negatives –  cryptic rearrangements missed that lead to PML-RARA fusion Rare small fusions can be missed Does not provide the sensitivity required for monitoring minimal residual disease (MRD)
Molecular Studies (Reverse Transcriptase-PCR) Allows for accurate quantitation of PML-RARA for monitoring response to therapy ARUP available tests PML-RARA Translocation, t(15;17) by RT-PCR, Quantitative 2002871	Test Characteristics Can detect all three PML-RARA fusion types  (long, short, and variable forms) Advantages Highly sensitive, specific, and quantitative Disadvantages False negatives – due to poor RNA yield May require more time than FISH testing
Cytogenetic (Conventional Karyotyping) ARUP  available tests Chromosome Analysis, Bone Marrow 2002292	Test Characteristics Uses G-banded metaphases from bone marrow culture Translocations (fusions) Classic translocation  t(15;17)(q22;q21) – PML-RARA Less common translocations t(11;17)(q23;21) – PLZF-RARA t(11;17)(q13;21) – NuMA-RARA t(5;17)(q35;21) – NPM1-RARA Advantages Highly specific Can characterize cases lacking the PML-RARA fusion Can detect rare subtypes of APL (eg, t(11;17), t(5;17)) Disadvantages Requires up to 10 days for results Requires good quality metaphases False negatives – cryptic rearrangements leading (PML-RARA fusion will be missed)
Sanz, M 2009; Sun T, 2008; Swerdlow  SH et al 2008; Zhou Y, 2012 Atlas of Genetic and Cytogenetics in Oncology 11-14-12

Cytogenetically-Normal AML (CN-AML) Prognostic Markers

Cytogenetic analysis is the initial test for risk stratification in acute myeloid leukemia (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.

Molecular Markers in CN-AML

Molecular Markers in CN-AML
Marker	Biology	Prognosis
Well-Characterized Markers
NPM1 mutation ARUP available tests: Whole blood or bone marrow	Nucleolar phosphoprotein that shuttles between nucleus and cytoplasm	NPM1 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 ARUP available tests: Whole blood or bone marrow	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 cytoskeleton	Overexpression associated with unfavorable outcome
MLL partial tandem duplication (PTD) mutations, overexpression	Involved in chromatin modification	MLL 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 AML and acute lymphoblastic leukemia (ALL)
    • 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/or FISH testing
    • SNP microarray cannot detect t(15;17) (PML-RARA), t(8;21) (RUNX1T1-RUNX1), or 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

Differential Diagnosis

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

Monitoring

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 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

• 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
  • 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 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) in acute promyelocytic leukemia with t(15;17)
  • Leukostasis associated with WBC >100x10⁹/L
    • Dyspnea, chest pain and headache
    • Oncologic emergency

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

Click the plus sign to expand the table of additional tests.

Test Name and Number	Comments
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
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
IDH1 R132H Point Mutation with Interpretation by Immunohistochemistry 2007357 Method: Immunohistochemistry	Sequencing is preferred