Acute Myeloid Leukaemia

Definition
Clinical Features
Laboratory Diagnosis

Morphology
Cytochemistry
Morphology and Cytochemistry by AML Subtype
Trephine Biopsy Histology
Flow cytometry
Immunohistochemistry/Immunofluorescence
Cytogenetics
Molecular Genetics

Classification
Cytogenetically Defined Subtypes
Treatment and Prognosis

DEFINITION

Acute Myeloid Leukaemia (AML) is currently defined by the FAB criteria. The percentage of blasts, the presence of cytochemical myeloperoxidase, the major cell types present defined by morphology and esterase cytochemistry, and the immunophenotype define the 8 FAB subtypes:

M0-M5: >30% myelo/monoblasts by morphology or immunophenotype.

M6: >50% erythroid precursors, >30% blasts in non-erythroid cells.

M7: >30% megakaryoblasts present.

Note: Cytogenetics, molecular genetics, previous MDS or MPD, previous chemo- or radiotherapy and the presence of trilineage myelodysplasia do not have any place in the FAB system. All these features which contribute to the definition of prognostically important sub-groups will be included in a new classification system currently under consideration.

CLINICAL FEATURES

Increasing incidence with age. Median age at diagnosis is 50 years.
Symptoms are due to marrow failure (anaemia, infection, haemorrhage) or hyperleucocytosis.
Rarely presenting symptoms are due to chloromas or CNS involvement.

LABORATORY DIAGNOSIS

MORPHOLOGY

Myeloblasts are usually medium sized with an eccentric nucleus and open chromatin. Cytoplasm shows variable basophilia. A few granules or Auer rods may be present.
Maturing granulocytes may be normal or show the dysplastic features seen in MDS.
Monoblasts are generally very large with abundant grey-blue or basophilic cytoplasm. Nuclei are round or lobulated, and are central in the cell. Fine azurophil granules may be present but Auer rods are rarely, if ever, present.
Promonocytes and monocytes show abnormal nuclear maturation, granulation and loss of basophilia.
Erythroid precursors may be normal or show varying degrees of dyserythropoiesis.
Eosinophils may be present in varying numbers, with normal or abnormal morphology. Specific abnormal appearances are linked with cytogenetic abnormalities and are described below.
Basophils are rarely increased in AML, and if present show abnormal or poor granule formation and nuclear maturation.
Megakaryocytes may be reduced or increased. Dysplastic hyperlobated, hypolobated, multinuclear, small and blastic forms may be present.

CYTOCHEMISTRY

Myeloperoxidase (MPO) identifies blast cells as myeloid. Auer rods are detected twice as frequently as on Romanowsky stains. Dysplastic neutrophils may be negative. Eosinophil granules are always positive. Monoblasts and promonocytes may be negative. Sudan Black B gives identical results.
Chloroacetate esterase (CAE) specifically identifies cells of the granulocyte lineage, from the early promyelocyte stage to mature neutrophils.
alpha-Naphthyl Acetate esterase (ANAE) stains monocytes and megakaryocytes at all stages of maturation.
Toluidine blue stains the granules of basophils and mast cells.
Periodic Acid Schiff (PAS) stains many cell types. It is not lineage specific but the pattern of staining may be helpful, e.g. positive NRBC, megakaryoblasts.

TREPHINE BIOPSY HISTOLOGY

Helpful when there is severe cytopenia and a dry tap, e.g. in megakaryoblastic AML.
Useful for identifying megakaryocyte dysplasia and dyserythropoiesis.
Immunophenotyping is possible when the diagnosis is in doubt.
Not suitable for fine classification which is based on percentages of cell types and cytological detail.

FLOW CYTOMETRY

Myeloid blasts express combinations of CD34, CD13, CD33, CD117, HLA-DR, CD14, CD15. Glycophorin A and CD42b identify erythroid and megakaryocyte lineage blasts respectively.
Essential to separate undifferentiated AML (M0) from ALL. May need supplementation with APAAP staining for cytoplasmic immunoreactive MPO.
Essential for diagnosing biphenotypic leukaemias and detecting aberrant (promiscuous) antigen expression.
Can identify patient specific unique blast cell phenotypes.
Can identify blasts of megakaryocyte, erythroid, monocyte and granulocyte lineages.
Can identify leukaemic contamination of 'remission' marrow harvests.
Can identify early relapse.

IMMUNOHISTOCHEMISTRY/IMMUNOFLUORESCENCE

APAAP staining for cMPO, cCD3 and cCD79 should be done in all cases of undifferentiated acute leukaemia.
Immunofluorescent staining for nuclear PML protein should be done in all cases of suspected acute promyelocytic leukaemia. A microparticulate pattern is diagnostic of APML.

CYTOGENETICS

An abnormal karyotype is found in 50-60% of AML cases at presentation.
t(15;17), t(8;21) and inv/del/t(16) are associated with specific morphology, younger patients and a good prognosis.
Complex karyotypes and abnormalities of chromosomes 5 and 7 are associated with older patients, trilineage dysplasia and a poor response to treatment.
Other karyotypic abnormalities are prognostically neutral.

MOLECULAR GENETICS

The 3 major good prognosis translocations are all detectable by RT-PCR, which can be used to detect up to 40% more cases than are found by metaphase cytogenetics.
All translocations with cloned breakpoint genes are theoretically detectable by RT-PCR.
Real time multiplex PCR may become available for routine diagnosis.
RT-PCR is used for monitoring speed of response to treatment and may be predictive for relapse.

FAB CLASSIFICATION OF THE ACUTE MYELOID LEUKAEMIAS

FAB TYPE	BLAST CELLS	MATURING CELLS	IMPORTANT DIAGNOSTIC TESTS
M0	<3% MPO positive blasts.	Dysplastic mature cells may suggest the diagnosis, but the blasts are undifferentiated by morphology and cytochemistry.	MPO, cMPO by immunocytochemistry, flow cytometry.
M1	>90% blasts in non-erythroid cells (NEC). >3% MPO or SBB positive.	<10% maturing granulocytes and maturing monocytes.	MPO/SBB, CAE, ANAE.
M2	Blasts 30-89% of NEC.	>10% of NEC are maturing granulocytes. <20% monocytic cells.	CAE and ANAE to show granulocyte and monocyte components.
M3	Granular or hyper-granular promyelocytes. Bundles (faggots) of Auer rods. Bilobed nuclei.	The neutrophils are often normal, occasionally dysplastic. Erythroid precursors and megakaryocytes do not show significant abnormalities.	Cytochemistry: Heavy MPO or SBB staining, strong CAE positivity. Immunofluorescence for PML nuclear protein. Cytogenetics: RT-PCR for t(15;17) and PML/RARa transcripts.
M3V	Promyelocytes are agranular with deeply basophilic cytoplasm. Bilobed nuclei are a prominent feature.		Cytochemistry, cytogenetics and molecular studies as for classical M3. The PML nuclear protein pattern is confirmatory and quick to do.
M4	Mixture of smaller myeloblasts and larger monoblasts.	>20% cells in the granulocytic lineage. >30% cells in the monocytic lineage.	CAE and ANAE stains to confirm cells of both granulocyte and monocyte lineages are present.
M4Eo	Characteristic large cells with large monocytoid nuclei and both eosinophil and large purple/black granules are present.	The proportion of granulocytic and monocytic cells are highly variable. Some cases are actually M2 by strict FAB criteria, but the characteristic 'eosinobasophils' are always present in varying numbers. May be scanty.	CAE and ANAE to identify the granulocyte and monocyte components. The abnormal eosinophil granules are also CAE positive. Cytogenetics and RT-PCR to confirm inv/del/t(16).
M5A/M5B	Large monoblasts and promonocytes with abundant cytoplasm.	M5A is predominantly monoblastic. M5B shows maturation with clear progression to promonocytes and monocytes which are dysplastic. Granulocytes <20% of cells.	CAE and ANAE. The CAE confirms the absence of any significant granulocyte component.
M6	>50% of all cells are erythroid precursors. >30% of the NEC are non-erythroid blasts.	Trilineage dysplasia is usually present. MDS is the main differential diagnosis.	The non-erythroid blasts may be MPO/SBB negative. Flow cytometry for CD34⁺ cells.
M7	AML with >30% megakaryoblasts. Often difficult to aspirate.	Micromegakaryocytes or abnormal more mature forms may suggest the diagnosis. The only form of AML where the trephine biopsy may be diagnostic.	Flow cytometry or immunohistochemistry to identify platelet specific antigens on the blasts.

Morphology and Cytochemistry by AML Subtype

AML-M1	AML-M2

Left: myeloblasts with typical eccentric nuclei. Auer rods visible. Right: Sudan black B staining and Auer rods.	Typical myeloblasts with eccentric nuclei and maturing dysplastic granulocytes at all stages of development.	Sudan black B positivity in blasts, stronger in dysplastic metamyelocytes. Inset: Chloroacetate in maturing granulocytes.
AML-M3

Hypergranular promyelocytes (right). Variable granule content and multiple Auer rods visible (left).	Sudan black B stain. Typical heavy cytoplasmic positivity.	Promyelocyte chloroacetate esterase positivity (blue). Inset: atypical ANAE positivity seen in 1% of M3 cases.
AML-M3v	AML-M4	AML-M5

Typical bilobed nuclei and basophilic cytoplasm packed with granules too small to resolve by light microscopy, but which give typical M3 cytochemical reactions.	Mixed population of myeloblasts and monoblasts. Inset: chloroacetate esterase positive granules (blue), ANAE-positive monocytes (green/brown).	Left: large monoblasts with central nuclei and abundant cytoplasm. Right: monoblast ANAE positivity (brown), and one blue chloroacetate esterase positive myelocyte.

AML-M6

Marked dyserythropoiesis (>50% of cells), with dysplastic granulocytes.	Left: giant multinucleate late normoblasts. Right: PAS stain; granular in proerythroblasts, homogeneous in normoblasts.
AML-M7

Megakaryoblasts / micro- megakaryocytes in bone marrow aspirate.	Trephine biopsy showing fibrosis, blast cells and atypical small megakaryocytes.

Future Developments

The classification and diagnostic criteria of the haematological malignancies, including Acute Myeloid Leukaemia, is currently under consideration and future developments may take account of known prognostic factors including cytogenetics, secondary AML and morphological assessment of dysplasia. New categories of AML may be required, as well as retention of elements of the FAB system. It is likely that RAEB-T will be incorporated into AML, i.e. the lower limit of blasts required for the diagnosis will be 20%.

The subtypes of AML defined by the t(8;21), t(15;17) and inv/del/t(16) are sufficiently distinctive to warrant recognition as separate categories. Cases with 11q23 abnormalities, although not correlated with specific morphology may be considered as a separate category.

Those cases evolving from myelodysplasia or post chemo- or radiotherapy can be grouped. They are linked by the presence of dysplasia and a poor prognosis. Apparent de novo cases with multilineage dysplasia may warrant recognition on the basis of their generally poor response to chemotherapy. For the remaining cases, some form of morphological classification will still be required. This is likely to be similar to the current FAB system, with the additional recognition of cases with basophil or mast cell involvement.

AML, predominantly granulocytic, with 3 abnormal basophils (arrowed) identified by their dense nuclear chromatin. Granules indistinct.

Toluidine blue staining (same case) showing 4 cells with prominent metachromatic staining of basophilic granules.

CYTOGENETICALLY DEFINED SUBTYPES OF AML

AML with t(8;21)

The morphology and cytochemistry of t(8;21) AML is characteristic.

The blast cells are variable in size, but have basophilic cytoplasm, a prominent Golgi region which abuts an indentation in the nucleus.
Long Auer rods are present in varying numbers.
Promyelocytes may show pseudo-Chediak large granules.
MPO and SBB staining shows strong positivity in all the blasts, localised to the Golgi region.
The maturing granulocytes beyond the myelocyte stage are agranular with flat peach coloured cytoplasm.
Nuclear maturation is defective, with frequent pseudo-Pelger bilobed nuclei.
One third of cases have increased eosinophils with abnormal granules; often very large, with variable amphophilic staining (blue/green and orange).
The proportion of blasts varies from <20% to >90%.
The majority of cases are M2, but 10-15% are M1, and 5-10% RAEB-T.


t(8;21). Typical basophilic myeloblasts with a Golgi region against the nucleus. Maturing granulocytes have empty pink cytoplasm. Auer rod visible in late metamyelocyte (centre left).	t(8;21). No maturation, but myeloblasts show typical Golgi region, often located in nuclear cup-like indentation (left). Sudan black B stain showing positivity localised to Golgi region (right).

t(8;21). Abnormal eosinophils (seen in 30% of cases) showing marked variation in granule size and staining.	t(8;21) M2 type. MPO (right) & Sudan black (left) showing intense localised positivity in blasts, heavier staining in more mature cells.

T(15;17) and ACUTE PROMYELOCYTIC LEUKAEMIA

In the classical form the majority of cells are granular or hypergranular promyelocytes.
Pathognomonic bundles or 'faggots' of multiple Auer rods are present, but may be rare.
The nuclei often show a distinctive bilobed or 'figure of eight' configuration.
MPO/SBB staining shows a heavy pattern, often completely filling the cytoplasm.
The maturation of the cells to the promyelocyte stage is confirmed by strong CAE staining, which may also stain the Auer rods.
The 'variant' hypogranular (microgranular) form shows the same characteristic bilobed nuclei, but the cytoplasm appears agranular and basophilic because the granules present are too small to be resolved by the light microscope.
Usually a few hypergranular cells are present to help suggest the diagnosis.
The cytochemical appearances are identical to the hypergranular form, although the CAE staining may be weaker.


t(15;17). Hypogranular variant of APML, showing typical bilobed nuclei and basophilic cytoplasm. Inset: high power magnification.	t(15;17). Composite illustration of the morphology and cytochemistry of APML.	Immunofluorescence stain for nuclear PML protein. Four nuclei show profuse microparticulate dots diagnostic of APML, and 1 nucleus (left) shows the normal pattern of a few large discrete dots.

Inv/del/t(16)

This subtype of AML is characterised by a large distinctive cell with unusual granules.
Abnormal small eosinophil granules co-exist with very large purple/black basophil-like granules.
The nuclei in these cells are large and monocytoid.
These cells have been called 'eosinobasophils' or 'hybrid' cells.
The background AML is also usually composed of large blasts and maturing cells.
Most cases fit the criteria for M4, hence the FAB title of M4Eo. However, the distinctive cells and the chromosome abnormality occur in cases that are clearly M2.
More mature eosinophils lose the purple granules and may appear fairly normal.
There is usually severe granulocyte and monocyte dysplasia, but trilineage dysplasia is not seen.


inv(16). Three large monocytoid 'eosinobasophils' with large purple/black granules and variable numbers of small poorly staining eosinophil granules.	inv(16). One typical large 'eosinobasophil', among a mixed population of myelo/monoblasts. One less florid typical cell at upper left.

TREATMENT AND PROGNOSIS

Intensive combination chemotherapy induces remission in >90% of children, >80% of adults below the age of 40, and >70% of adults aged 40-55 years. Thereafter the the remission rate drops steeply with age.
Options after remission induction are further consolidation therapy, followed by a high dose therapy and autologous stem cell rescue, or allogeneic BMT.
In adults under the age of 55 years 5 year disease free survival is now approximately 35%. Patients who have one of the three cytogenetically defined good prognosis subtypes have a 5 year disease free survival of approximately 50%.

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M0-M5:	>30% myelo/monoblasts by morphology or immunophenotype.
M6:	>50% erythroid precursors, >30% blasts in non-erythroid cells.
M7:	>30% megakaryoblasts present.


AML, predominantly granulocytic, with 3 abnormal basophils (arrowed) identified by their dense nuclear chromatin. Granules indistinct.	Toluidine blue staining (same case) showing 4 cells with prominent metachromatic staining of basophilic granules.