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Diagnosing Acute Myeloid Leukemia


Medical editor Lorentz Brinch MD
Hematologist
Oslo University Hospital

General

Acute myeloid leukemia is divided into 7 subgroups (M0-M7). The group division is based on the morphological examination after the FAB-classification of blood and bone marrow smears and can be supported by immunophenotyping, cytogenetic, and molecular genetic testing, as well as combinations of these techniques. This is done in the WHO staging which is increasingly applied also in Norway.

It is important to obtain a diagnosis and determine which subgroup the patient belongs to. The more rare subgroup, M3 (acute hypergranular promyelocyte leukemia), is especially important to identify as it is often accompanied by non-compensated disseminated intravascular coagulation (DIC). This type of leukemia requires special treatment which must be started immediately when there is a well-founded suspicion of this diagnosis. With correct treatment the prognosis is far better than for the other subgroups.

The type of chromosome changes in leukemia cells at the time of diagnosis may be of importance for treating patients, especially those under 60 years. Patients are divided according to low and high risk criteria.

Indication

  • Suspicion of acute myeloid leukemia 

Goal

  • Confirm the diagnosis
  • Identify risk group for disease progression
  • Cure the disease

Norsk selskap for hematologi. Akutt myelogen leukemi, handlingsprogram [Online]. Februar 2007 [hentet 15. april 2007]; tilgjengelig fra: URL: http://www.legeforeningen.no/asset/15380/1/15380_1.doc


Examinations

Bone marrow aspiration

A bone marrow aspiration is necessary to diagnose acute myeloid leukemia.

Examinations performed on the aspirate:

  • Microscopy, MGG staining and possible cytochemical staining
  • Immunophenotyping (flow cytometry)
  • Cytogenetics with directed testing using FISH or molecular genetic techniques
  • Vital freezing of leukemia cells and/or RNA/DNA extraction for later molecular genetic analyses should be done, if possible.

Biopsy of the iliac crest is not obligatory unless the aspirate is inadequate. The biopsy can in some cases provide additional valuable information. 

Blood tests

General:

ABO typing, hemoglobin, leukocytes with differential counting, thrombocytes, blood smear, LD, albumin, creatinine, Na, K, Ca, phosphate, urate, glucose, CRP, bilirubin, AFOS, ASAT, ALAT, LDH, CMV.

Special testing:

  • HLA typing – taken if there is a need for HLA compatible blood components
  • Lysozyme – in case of raised lysozyme it is probable the patient has acute myeloid leukemia
  • FLT3 analysis (PCR) – a prognostic factor which indicates high risk of recurrence

Lumbar puncture

A lumbar puncture is performed when there is a clinical suspicion of CNS leukemia. Patients with M4-M5 with high LPK (leukocyte particle concentration) have an increased risk for spreading to the central nervous system.

A cytological examination of the aspirate is performed and possibly with immunophenotyping, cell counting and measurement of spinal protein. In case of elevated cell count, cytospin with MGG staining is performed.

Interpretations of findings in spinal fluid can be difficult as there may be contamination with blast containing blood during the spinal puncture.

Familial evaluation

In cases where an allogeneic stem cell transplant is considered, HLA typing of siblings and parents is done when remission is achieved. In a clinical situation, this means all patients under 60 who reach complete remission.

It is important to emphasize that this does not necessarily mean that there will be indication for allogeneic stem cell transplantation in all cases. This will depend on a number of prognostic factors, comorbidity and the course of the disease during and after the induction therapy.


Definitions

May-Grünwald/Giemsa staining

In order to differentiate between cell structures, the May-Grünwald/Giemsa stain is used (MGG stain). This staining method works by a chemical reaction between stain substances and the biochemical components of the cell. The stain substances are neutral salts which ionize in water and react with oppositely charged ions in the cell. MGG staining is the basis for the FAB classification. 

Alpha-naphthyl acetatesterase staining

In many cases of MGG stained of bone marrow specimens, and even easier in peripheral blood, there may be indications of myelomonocytic leukemia (FAB classification M4-M5). In certain cases, these immature cells will have an abundance of cytoplasm, but no apparent clover leaf-cuts in nuclei, making the FAB classification ambiguous (either M1-M2 or M4-M5). In these cases, the alpha-naphthyl acetatesterase stain is useful, which is positive in myelomonocytic leukemia and monocytic leukemia and negative for other types.

Diaminobenzidine peroxidase staining

Of the cytochemical examinations, diaminobenzidine peroxidase (DAB stain) is the most useful supplement to MGG staining. Some acute myeloid leukemia, as when using the MGG stain, have under 5% promyelocytes and will have more than 5% granulated, immature/blast-resembling cells when using this stain. 

Lysozymes in serum

The lysozymes are produced by cells in the granulocytic series and predominantly the monocytic series. The enzyme is not produced by cells in the lymphocytic series. If a patient with acute leukemia has a raised serum lysozyme, it is probable the patient has acute myeloid leukemia and acute lymphatic leukemia is very unlikely. If the serum lysozyme is more than 4 times over the normal limit, this indicates M4 or M5.

Flow cytometry immunophenotyping

Immunologically, the disease is defined by expression of two or more of the following myelomonocytic markers: myeloperoxidase (MPO), CD13, CD33, CD65 and/or CD117. MPO is considered to be the most specific myeloid marker. An attempt at correlating immunophenotype with subtypes with regard to the FAB classification has been made, but there are only 3 subtypes which are clearly defined by their immunophenotype: M0, M6 and M7.

Cytogenetic and molecular genetic testing

Using genetic technology methods, for example FISH analysis, (fluorescent in situ hybridization) or PCR (polymerase chain reactions), details are revealed of gene material which change places, lacks or changes of chromosomes, and the significance this has for differentiation and growth of leukemia cells. This has an increasing significance for treatment since the methods are very sensitive for the presence of characteristic gene material from leukemia cells. An example of this is testing for internal tandem duplication (FLT3 gene) which is an unfavourable prognostic sign. 

FLT3 is a tyrosine kinase receptor which has a large impact on hematopoetic cell proliferation. In 18-30% of all adult patients, an internal tandem duplication (ITD) is observed of FLT3-ITD, which is considered to cause an increase of tyrosine phosphorylation leading to leukemia development.

The FLT3 mutations occur most often in acute myloid leukemia with normal karyotype and high blast value in the blood. There is agreement that FLT3-ITD is associated with high recurrence risk in younger patients. The FLT3 gene is also observed in M3,but here without indicating a poorer prognosis.

Another example is nucleophosmin1 mutation (NPM1-mutation). In patients with normal cytogenetics and absence of FL3T-ITD the presence of such mutations is associated with better than average prognosis.

Gene technology methods are increasing the understanding of leukomogenesis and thus contributing to development of new treatment principles. Testing for the APL/retinol alpha receptor, the translocation with DNA-hybridization techniques in M3, is an example of this. For this type of leukemia, very good results are achieved by treatment with retinol, which causes differentiation and apoptosis of leukemia cells.

More than half of the patients with acute leukemia have non-random (clonal) chromosome changes. That is, the same change in two or more mitoses from bone marrow cells. Certain cytogenetic changes are associated with special morphological changes.

Great emphasis is put on the results of such examinations during the risk consideration regarding transplantation of allogeneic stem cells in the first remission.


Implementation

The most important morphological finding during microscopy of newly discovered leukemia is evidence of Auer rods in cytoplasm of leukemia cells. The finding is characteristic for acute myeloid leukemia but is only found in 30% of patients.  

Typical findings in acute myeloid leukemia

Immature M1 / sign of maturition M2  / hypergranular promyelocyte M3

  • Auer rods (the minority of patients have Auer rods in leukemia cells)
    • In M3, many large Auer rods are often observed. These patients often have disseminated coagulation (DIC). The promyelocytes are often the most heavily granulated.
  • ≥ 5% promyelocytes
  • Peroxidase positive
  • Possibly immunophenotype, but this is usually unnecessary 

M3 is important to recognize since early initiation of treatment may lead to a relatively good prognosis in this subgroup.

In some patients with FAB type M2, the translocation t(8;21) is present. For M3, most of the translocations found are at t(15;17). Patients with these cytogenetic changes have a relatively good prognosis.

Myelomonocyte M4 / monoblast M5

  • Auer rods (minority of patients with AML have Auer rods in leukemia cells)
  • a-naphthylacetatesterase positive
  • Raised lysozyme
  • Possibly immunophenotype, but this is usually unecessary

Inversion of chromosome 16 is observed in myelomonocyte leukemia (M4) with abnormal eosinophils in bone marrow. These patients have a relatively good prognosis.

Erythro M6

Characteristic findings of bone marrow are >30% blasts of non-erythroid cells > 50 % erythropoiesis, most often dyserythropoiesis.

If there is suspicion of erythrocyte leukemia (M6), it is important to be certain the patient does not have megablastic anemia as a result of a B12 or folic acid deficiency or dyserythropoietic anemia.

Undifferentiated M0 / megakaryoblastic M7

  • Immunophenotype

To settle for a diagnosis for acute M0 and M7, it is necessary to carry out immunophenotyping with flow cytometry and/or immunohistochemistry.


Follow-up

As soon as the diagnosis is made, treatment is initiated.

Prognosis grouping

Low risk

Low risk patients have a favorable chromosome abnormality such as Inv(16), t(8;21) and t(15;17). An allogeneic stem cell transplantation is usually not appropriate for these patients in their first remission. NPMI mutation with normal cytogenetics and absence of FLT3ITD.

High risk

An unfavorable deviation may be an indication to start searching for unrelated stem cell donors early in the disease course, if the patient does not have a familial donor.

High risk criteria for AML
Late remission > 1 induction course
Multiple cytogenetic defects > 5
Cytogenetic abnormality Inv (3), t(3;3), -5, -7, part 5, part 7, t(9;22),
Secondary AML Earlier chemotherapy and/or myelodysplastic syndrome
Internal duplication of FLT3 gene  

All others are considered standard risk.

In all patients where there is a reason to start intensive inductive treatment, the cytogenetic examination of bone marrow cells should be done at the time of diagnosis before start of treatment. This result is decisive for prognosis grouping which is central for the possible indication for an allogeneic stem cell transplantation (STC) early in the disease course. 

In patients over 60 years, the result may be a guide for the intensity of the treatment which must be given when remission is achieved after induction treatment. Older patients with a cytogenetic abnormality indicating a poor prognosis hardly benefit from additional intense consolidation treatment, which causes bothersome or life-threatening complications, and little to no improvement in long-term prognosis. It is important not to over-treat the patient.  

Results from cytogenetic examinations must be available when the patient is ready for consolidation treatment after induction treatment.


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