At a Glance

Acute myeloid leukemia (AML) is an acquired hematologic malignancy that results in a clonal expansion of immature myeloid cells (myeloblasts or promyelocytes) in the blood or bone marrow and, less frequently, as accumulations of blasts in the peripheral tissues. Blasts in the bone marrow or peripheral blood must exceed 20% of nucleated cells, unless specific cytogenetic aberrations are identified. AML is predominantly a disease of adults and has been increasing in incidence as the population ages and as there are increasing numbers of survivors of childhood/young adult malignancies treated with cytotoxic agents. In 2010, the incidence of AML was 12.330, with the vast majority of patients dying from their disease.

Patients present with AML in a variety of fashions. The most common presentation is with symptoms related to cytopenias. Because of overgrowth of the marrow by the leukemic clone, patients can suffer weakness and fatigue from anemia, infections from neutropenia, or bleeding complications, such as gingival bleeding, petechiae, ecchymosis, epistaxis, or menorrhagia, from thrombocytopenia. Less commonly, AML can present with signs and symptoms of extramedullary disease, including skin, gingival, or CNS lesions. In patients with acute promyelocytic leukemia (APL), a specific morphologic and genetic subtype of AML, patients can present in life-threatening disseminated intravascular coagulopathy (DIC), which is a medical emergency.

The diagnosis for AML typically begins with the identification of increased blasts or promyelocytes in the peripheral blood, which are out of proportion to any left-shift in maturation or with a hiatus in maturation between the leukemic cells and more mature circulating myeloid elements (bands and neutrophils). When blasts comprise less than 20% of circulating nucleated cells, the peripheral blood count alone is insufficient for the diagnosis of AML.

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What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?

The work-up for AML achieves 2 main purposes. The first is to diagnose and characterize the disease, including identifying prognostic markers, markers to monitor disease, and to guide risk-adapted therapeutic strategies. The second is to gauge the patient’s ability to tolerate the potential therapeutic regimens.

Initial work-up

Initial work-up to diagnose AML, beyond a full history and physical exam, should include a complete blood count (CBC) with differential, a chemistry panel, and prothrombin time/partial thromboplastin time (PT/PTT)and fibrinogen. The latter coagulation tests are critical for recognizing evolving DIC associated especially with APL. In addition, if the patient is suffering any neurologic symptoms, a computerized tomography (CT) or magnetic resonance imaging(MRI) study and lumbar puncture with cytology of the cerebral spinal fluid (CSF) are required to stage the disease.

Bone marrow blast qualifications

The key diagnostic tests depend on the acquisition of a bone marrow specimen. Bone marrow is required for an accurate assessment of the degree of involvement. The leukemic cells are quantified by both morphology and flow cytometry. Often, there are morphologic features of the leukemia cells that can give clues to the subtype of AML, as well as help distinguish leukemic blasts from physiologic blasts during future monitoring.


With the exception of the pathogenomonic finding of Auer rods, the nature of the blasts must be proven to be myeloid by cytochemical stains and/or immunophenotyping. Cytochemical stains, such as myeloperoxidase (MPO) and non-specific esterase (NSE), are commonly used to establish a myeloid or monocytic lineage of the blasts. However, immunophenotyping, typically performed by flow cytometry, is much more widely used in most current laboratories to determine lineage and is critical in the cases of MPO-negative AMLs (AML M0 using the former French-American-British (FAB) classification system or undifferentiated AML). At least 2 myeloid markers must be positive (typically with <2 aberrant lymphoid markers) to indicate myeloid lineage.

MPO can also be examined by flow cytometry. Immunohistochemical (IHC) stains can be utilized if the marrow is inaspirable, although it is less quantitative than flow cytometry and there are fewer available lineage specific IHC stains. Although most practicing hematologists follow the 2008 World Health Organization (WHO) classification scheme, some still describe the leukemic cells using the former FAB classification system (AML M0-M7) for which immunophenotyping and cytochemical stains play key roles in determining the appropriate subtype.

Metaphase cytogenetics

A bone marrow biopsy is also required to identify any recurrent cytogenetic studies that are critical to the diagnosis of AML according to the WHO. Seven different recurrent genetic abnormalities currently merit their own diagnostic category in the 2008 WHO classification system and carry associated prognostic and therapeutic implications. These are: t(8;21), t(15;17), inv(16) or t(16;16), t(9;11), t(6;9), inv(3) or t(3;3), and t(1;22). The first 3 categories carry a better overall prognosis, whereas the remaining 4 are adverse prognostic markers. A normal karyotype (NK-AML) is considered of intermediate prognosis overall. Myelodysplasia-related AML is also a specific recognized subtype of AML and may be characterized by cytogenetic abnormalities seen in myelodysplastic syndrome (MDS).


On occasion, fluorescent in situ hybridization (FISH) for recurrent AML translocations or changes common in MDS can be helpful markers for disease monitoring or allow for rapid diagnosis, especially if APL with t(15;17) is suspected. Emergent recognition of t(15;17) is required for timely initiation of potentially life-saving all-trans-retinoic acid (ATRA).


The t(15;17) translocation can also be emergently identified by quantitative reverse transcription polymearase chain reaction (RQ-PCR). In addition, RQ-PCR on the bone marrow aspirate is required to establish a baseline for monitoring therapy in t(15;17) or t(9;22) positive AMLs (the latter in cases of transformation from chronic myelogenous leukemia; CML). Reverse transcription PCR can also be used to identify many of the recurrent translocations in a qualitative manner.

Other molecular tests

In addition, molecular tests must be performed on the diagnostic sample. These carry significant prognostic implications and may represent therapeutic targets in the future as well. Although there are many potential molecular mutations that have been suggested to carry prognostic implications in AML, the clinical significance of most are still under investigation. However, FLT3, NPM1, and CEBPA mutations have come into regular clinical practice and c-KIT mutations are assessed by a subset of clinicians. The import of these molecular markers is such that NPM1 and CEBPA mutations in NK-AML comprise their own provisional diagnostic categories of AML. FLT3, while not determining a specific AML subtype, is a critical prognostic modifier across all subtypes of AML.

FLT3 encodes a receptor tyrosine kinase that plays a critical role in proliferation, differentiation, and survival in hematopoietic cells. It is mutated in 25-30% of AMLs. The majority of these are internal tandem duplications (FLT3-ITD, 20% of AMLs), whereas the remaining are mutations in the tyrosine kinase domain itself. Although the latter are of unclear clinical significance, FLT3-ITDs carry a poor prognosis, especially in the 28-34% of NK-AMLs. In addition, FLT3-ITDs are common in APLs and negate the favorable outcome associated with t(15;17) translocation. Many laboratories offer FLT3-ITD testing by PCR, which can detect the approximate 70% of ITDs that occur in the juxtamembrane region. PCR is quite sensitive and can detect as few as one FLT3-positive cell in 10,000-1,000,000 cells. Testing is appropriate in all new diagnoses of AML.

NPM1 encodes a nuclear-cytoplasmic shuttle protein abnormally localized in the cytoplasm of NPM1-positive AMLs because of the presence of 4-bp insertions located in various locations in the gene. These mutations are found in 25-35% of AMLs, including 45-64% of NK-AMLs. When mutation is positive, patients with NK-AMLs have significantly improved overall survival, and younger adult patients may, therefore, not gain any benefit from allogeneic stem cell transplant (SCT) in first complete remission (CR1). Testing for NPM1 4-bp insertions, performed by PCR, is widely available and appropriate in NK-AMLs.

Like NPM1, CEBPA mutations confer a better prognosis for patients with NK-AML. CEBPA encodes a transcription factor important in hematopoiesis that can acquire mutations in either its N or C termini. Only biallelic mutations that involve both N and C mutations (occurs in 90% of ciallelic cases) are associated with favorable outcomes. Since the location of the mutations range throughout the entire length of the gene, testing for CEBPA is typically performed by sequencing and, therefore, may require up to 40% neoplastic cells for accurate detection of the mutations. Testing is appropriate in NK-AMLs.

AMLs that contain reciprocal translocations involving core binding factor genes, t(8;21) and inv(16)/t(16;16) (involving CBFα and CBFβ, respectively, the CBF-AMLs), are also tested for the presence of c-KIT mutations. The most common mutations cluster in exons 8 and 17, but only those in exon 17 have been found to confer an unfavorable prognosis when found in CBF-AMLs.

Ideally, testing for c-KIT mutations would be performed only after the diagnosis of a CBF-AML had been made. Similarly, NPM1 and CEBPA testing should only be performed in NK-AMLs. However, since cytogenetic results may take several days to obtain and since, for most clinicians, these tests are not available at their own institutions, typically these tests are sent to reference labs at the time the diagnostic material is obtained without waiting for cytogenetic results. Although many unnecessary tests are, therefore, performed, this practice ensures that important prognostic and disease monitoring markers are identified at diagnosis and are available for decisions regarding allogeneic SCT. As inhibitors of some of these targets become increasingly available, these results may also be of direct therapeutic import.

Additional tests

Additional tests to assess a patient’s ability to tolerate chemotherapy are required. Most AML induction regimens include either daunorubicin or idarubicin. These anthracyclines carry a significant risk for cardiotoxicity. Thus, a baseline echocardiogram and electrocardiogram (EKG) are important tests to order.

Additionally, histocompatibility antigen (HLA) typing should be performed to prepare for the exigency of allogeneic stem cell transplantation (SCT) in many AML patients. In patients with high risk features (i.e., high white blood cell count, molecular markers of poor prognosis, high risk cytogenetic abnormalities) without sibling donors, an alternative donor search may also begin at diagnosis.

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

Other factors that may affect laboratory results include the adequacy of the bone marrow sample, especially if an inadequate bone marrow biopsy is performed that does not reflect the bone marrow contents. Although the aforementioned studies can be performed on the peripheral blood, a bone marrow sample is required for evaluating the extent of marrow disease and assessing marrow cytogenetics. Of note, AML can still be diagnosed if blasts are fewer than 20% of nucleated cells if associated with t(8;21), t(15;17), or inv(16)/t(16;16) cytogenetic changes.

Cytogenetic studies and molecular studies require specific preservatives, heparin for the former and ethylenediaminetetraacetic acid (EDTA) for the latter. Selecting the wrong tube type can preclude the requisite testing. In addition, inadequate sampling can fail to provide sufficient specimen for testing. In particular, CEBPA testing performed by DNA sequencing requires significant sample volumes, often as much as 7 mL of bone marrow aspirate.

What Lab Results Are Absolutely Confirmatory?

The identification of t(8;21), t(15;17), or inv(16)/t(16;16) by cytogenetics or FISH [or RQ-PCR in the case of t(15;17)] is diagnostic of AML, regardless of the percentage of leukemic cells in the bone marrow. In all other cases, the identification of more than 20% leukemic cells in the peripheral blood or bone marrow is required for diagnosis of AML. This can be achieved by differential count of sample type, flow cytometry on either sample, or immunohistochemical stains on the bone marrow biopsy. The definition of leukemic cells denotes a clonal proliferation with a block in maturation. Increased blasts associated with growth factor stimulation or reactive conditions are not examples of leukemia.

What Tests Should I Request to Confirm My Clinical Dx? In addition, what follow-up tests might be useful?


Currently, only periodic bone marrow biopsy with quantification of blasts by differential count or immunophenotyping (most commonly by flow cytometry) and regular CBCs are used to monitor most patients with AML. A bone marrow study is performed 7-14 days after induction to document the extent of marrow cytoreduction and 4-6 weeks after induction to document marrow recovery.

Since the patient is often hospitalized for the duration of this time, daily CBCs are performed. Depending on the risk factors at diagnosis, most patients receive either consolidation therapy or proceed to allogeneic SCT in CR1 following consolidation therapy. CBCs are preformed every 1-3 months for 2 years and every 3-6 months up to 5 years. Bone marrow studies need only be performed if the patient develops cytopenias or if abnormalities are found on the peripheral blood smear. Many hematopathologists, however, perform bone marrow biopsies for reassurance yearly.

During these periods of follow-up, ancillary studies may be performed on the bone marrow to monitor the disease. Only RQ-PCR for t(15;17) and t(9;22) are strongly recommended to follow the disease. However, many clinicians follow the patients with cytogenetic studies to watch for any abnormalities found at diagnosis and to identify any new abnormalities that may suggest the development of a secondary clonal marrow disorder, such as a therapy-related myelodysplastic syndrome or clonal evolution of the original AML. However, cytogenetic studies have poor analytical sensitivity. In addition, molecular markers present at diagnosis may also be monitored with superior analytical sensitivity, except in the case of CEBPA (which has poor sensitivity and should not be used for minimal residual disease testing).

When there is a molecular marker to follow, FISH studies play little role in monitoring disease because of their typically lower sensitivity. Flow cytometry, when a characteristic immunophenotype profile is available, can also provide a useful measure of monitoring disease in AML patients. However, this method is limited by the absence of aberrant markers on some patient’s leukemic blasts and by the common phenomenon of immunophenotypc evolution in AML.

Are There Any Factors That Might Affect the Lab Results? In particular, does your patient take any medications – OTC drugs or Herbals – that might affect the lab results?

Errors in test selection for AML

If a bone marrow biopsy and aspirate are performed, it is unnecessary to perform the ancillary testing (flow cytometry, cytogenetics, molecular PCR, or RQ-PCR) on the peripheral blood as well; although sometimes when leukemic lineage is unclear, a limited flow cytometry panel may be helpful on the peripheral to guide testing on the subsequent bone marrow specimen.

In addition, if there is clear leukemia without any prior history of a myeloproliferative neoplasm, it is unnecessary to perform testing for JAK2 mutations. BCR/ABL1 testing is not typically performed on de novo AMLs, although rarely CML can present as an acute myeloid sudden blast crisis. However, in such cases, typically the t(9;22) can be identified by metaphase cytogenetic studies.

Errors in interpretation of test results for AML

Because of the range of mutations in the FLT3, NPM1, and c-KIT genes, allele-specific primers in PCR only detect the mutations they are designed to target. For these genes, limitations in clinical sensitivity can afford false negative results. For example, standard primers for FLT3 target the juxtamembrane region, but only 70% of ITDs are located in this region; therefore, 30% of ITDs will give a false negative result. Similar caveats hold for NPM1 and c-KIT, which can both have disease-associated mutations outside the commonly targeted primer sites. For CEBPA, false negatives can result because of problems of analytical sensitivity, since sequencing typically requires up to 40% leukemic cells for recognition of the mutations.

Sampling issues may also lead to errors in interpretation. A hemodilute specimen that does not sample the marrow contents may be spuriously negative by cytogenetics or molecular studies if the leukemic cells are not represented in the blood.