Acute myeloid leukemia

AML

1. Acute Myeloid Leukemia

2. Acute Myeloid Leukemia
- Also known as
•Acute myelocytic leukemia
•Acute myelogenous leukemia
•Acute nonlymphocytic leukemia
Stem cell disorder characterized by Clonal expansion of
myeloid precursor cells with reduced capacity to
differentiate i.e, MATURATION ARREST.

3. Common myeloid progenitor

4. Predisposing factors
Congenital factors :-
•Down syndrome.
•Bloom syndrome.
•Monosomy 7 syndrome
•Klinefelter syndrome
•Turner syndrome
•Neurofibromatosis
•Congential dysmorphic syndrome

5. Predisposing factors
Marrow failure syndrome:-
•Fanconi anemia
•Dyskeratosis congenita.
•Schwchman Diamond Syndrome
•Amegakaryocytic thrombocytopenia
•Blackfan Diamond syndrome
•Kostmann Agranulocytosis
•Familial aplastic anemia
•Familial platelet disorder.

6. Predisposing factors
Environmental factors:-
•Solvents ( benzene )
•Smoking
•Ionizing radiation
•Non ionizing radiation
•Chemotherapy
Alkylating agent
Topoisomerase II inhibitor

7. Biological features
•Leukemogenesis- result from block of differentiation as
well as altered proliferation and impaired apoptosis
through genetic dysregulation.

8. Genetic Associations
•Research states that AML is caused by genetic
aberrations such as translocations between
chromosomes that alter the function of transcriptory
regulatory factors
•These translocations are a direct result of chimeric
fusion proteins which are caused by the abnormal cells
and its inability to allow further growth, proliferation,
maturation and differentiation.
•Class 1 and 2: mutations responsible for the
development of the neoplastic process of
myeloproliferation and de-differentiation

9. Genetic Associations
•Class 1: mutations that give rise to proliferation and/or
differentiation.
•Class 2: mutations that interfere with terminal
differentiation and apoptosis thereby providing survival
advantage for the mutated cells.

11. Classification of AML
•WHO classification.
•FAB classification.

12. Differences between FAB and WHO
FAB-classification:
1)Heavily used “Morphologic Findings”
2)Special staining (SBB, MPO, NSE, etc), if required
WHO-classification:
1)Morphologic findings
2)Special staining (decreased role)
3)Immunophenotyping (in the form of FC and IHC) heavily used.
4)Cytogentics and Molecular genetics studies frequently used.

13. WHO Classification of Acute Myelocytic
Leukemias

14. FAB Classification of AML
•M0 undifferentiated acute myeloblastic leukemia (5%)
•M1 AML with minimal maturation (20%)
•M2 AML with maturation (30%)
– t(8;21)
•M3 Acute promyelocytic leukemia (5%)
–t(15;17)
•M4 Acute myelomonocytic leukemia (20%)
•M4 eos Acute myelomonocytic leukemia with eosinophilia (5%)
–inv (16)
•M5 Acute monocytic leukemia (10%)
–t(9;11)
•M6 Acute erythroid leukemia (3%) (DiGuglielmo's disease)
•M7 Acute megakaryoblastic leukemia (3%)

15. Clinical features
Due to Bone Marrow Failure
Due to Organ Infilteration
Others

16. History from patient with leukemia
Increasing fatigue or decreased exercise tolerance (anemia)
Excess bleeding or bleeding from unusual sites (DIC,
thrombocytopenia)
Fevers or recurrent infections (granulocytopenia)
Headache, vision changes, nonfocal neurologic abnormalities
(CNS leukemia or bleed)
Early satiety (splenomegaly)
Family history of AML (Fanconi, Bloom or Kostmann
syndromes or ataxia telangiectasia)
History of cancer (exposure to alkylating agents, radiation,
topoisomerase II inhibitors)
Occupational exposures (radiation, benzene, petroleum
products, paint, smoking, pesticides)

17. Physical Examination
Ecchymosis and oozing from IV sites (DIC, possible acute
promyelocytic leukemia)
Fever and tachycardia (signs of infection)
Papilledema, retinal infiltrates, cranial nerve abnormalities (CNS
leukemia)
Poor dentition, dental abscesses
Gum hypertrophy (leukemic infiltration)(M4)
Skin infiltration or nodules (leukemia infiltration)(M4)
Lymphadenopathy, splenomegaly, hepatosplenomegaly
Back pain, lower extremity weakness [spinal granulocytic
sarcoma, most likely in t(8;21) patients]

18. Acute Myeloid Leukemia:
Diagnostic Steps
1. Evaluation of an abnormal CBC for possible AML
•Confirm bone marrow failure, assess for blasts/blast equivalents and dysplasia
•WBC: non-specific; in AML can be low, normal, or high
•ANC: severe neutropenia characteristic of HP failure; typical in AML, but exceptions
occur
•Circulating blasts: variable number and percent in AML, but key feature to assess
in blood
•RBC features: severe anemia characteristic of HP failure, an expected feature of AML
•Polychromasia: reduced, since anemia is result of bone marrow production failure
•Other RBC pathology: non-specific
•Platelets: severe thrombocytopenia characteristic of HP failure

19. 2. Identify morphologic blasts and blast equivalents in blood (and
subsequent bone marrow, if performed)
•Morphologic assessment of nuclear features is key for “blast”
designation
A cell can be a blast if it is exhibiting finely dispersed rather than
condensed nuclear chromatin. Other useful nuclear features include
overall size, nucleoli and nuclear configuration.

20. •Cytoplasmic features are very helpful in lineage determination, ie,
sparse fine granules and Auer rods in myeloblasts, cytoplasmic
blebbing in megakaryoblasts, and deeply basophilic, vacuolated
cytoplasm in erythroblasts

21. Myeloblast
Relatively high nuclear / cytoplasmic ratio.
Finely dispersed chromatin and variably
prominent nucleoli
Variable number of cytoplasmic granules,
may be concentrated in limited portion of
cytoplasm

22. Promyelocyte
Nuclear chromatin slightly condensed;
Nucleoli variably prominent;
Nucleus often eccentric, and Golgi zone
may be apparent
Numerous cytoplasmic granules that may
be more dispersed throughout cytoplasm
Blast equivalent in APL only
In APL, intense cytoplasmic granularity
usually present .Nuclear configuration
variable, but nuclear folding and lobulation
characteristic of microgranular variant of
APL

23. Monoblast
•Moderate to low nuclear to
cytoplasmic ratio,
•Nuclear chromatin finely dispersed
with variably prominent nucleoli;
nuclei round to folded
•Abundant, slightly basophilic
cytoplasm containing fine granulation
and occasional vacuoles

24. Promonocyte
Slightly condensed nuclear chromatin;
Variably prominent nucleoli
Abundant finely granular blue/gray
cytoplasm that may be vacuolated
Very monocytic appearance with nuclear
immaturity
Consistent blast equivalent in AML

25. Erythroblast
Relatively high nuclear/cytoplasmic ratio
Nucleus round with slightly condensed
chromatin;
Nucleoli variably prominent
Moderate amounts of deeply basophilic
cytoplasm that may be vacuolated
Included in blast percentage only in acute
erythroid leukemia

26. Megakaryoblast
Highly variable morphologic features;
May be lymphoid-appearing with high
nuclear to cytoplasmic ratio
Nuclear chromatin fine to variably
condensed
Cytoplasm may be scant to moderate, is
usually agranular or contains a few
granules;
Blebbing or budding of cytoplasm may be
evident
Blasts may form cohesive clumps

27. MorphologyLymphoblast Myeloblast
Nuclear
chromatin
Coarse Fine
Nucleoli 1-2 3-5
N:C ratio High High
Auer rod Absent Present
Accompanying
cells
Lymphocytes Myeloid
precursor

28. 3. Bone marrow examination often performed to address
differential diagnoses from blood assessment or for protocol
requirements.
4. Enumeration of blasts/blast equivalents by morphology and differential
cell count
•Previously >30% blasts on BM aspirate (per FAB criteria)
•As per recent WHO criteria, AML is defined by greater than 20% blasts on
BM aspirate.
–patients with certain cytogenetic abnormalities are considered to have
AML regardless of blast percentage
•t(8;21)(q22;q22), inversion (16)(p13q22)
•t(16;16)(p13;q22), and t(15;17)(q22;q12)
Unique situations compromising blast count:
Fibrosis and/or necrosis
Predominance (≥50%) of erythroid lineage
Marked hypocellularity
Technically poor specimen

29. •5. Determine lineage of blasts/blast equivalents (can be performed on
blood or bone marrow)
Morphology (nucleus and cytoplasm)
Cytochemistry
Immunophenotype

30. Myeloperoxidase stain
•Basis- breakdown of hydrogen peroxide by enzyme
myeloperoxidase releasing an oxygen radical that reacts
with a soluble substrate to form colored precipitate.
• MPO located in peroxisomes of neutrophils and
monocytes and specific granules of eosinophils.
•Staining is more pronounced in golgi region.

31. MPO positive

32. Sudan Black B
•It is a direct stain phospholipid in granular membrane.
•Auer rods are MPO and SBB positive.

33. Esterase stains
•Non specific esterase reactivity is found in monocytes.
•Basis- Enzymatic release of a side chain from a naphthol
ring with subsequent reaction of the free ring with a
soluble colour develops to generate a coloured
precipitate.
•Most common used substrate for Non specific esterase
are Alpha- naphthyl butyrate and Alpha – naphthyl
acetate.

34. NSE positive in MONOCYTIC CELLS

35. PAS staining
•Periodic Acid Schiff stain reacts primarily with glycogen,
generating a fuchsian coloured precipitate.
PAS+ MEGAKARYOCYTES

36. AML – cytochemistry

Reaction

M0

M1 M2 M3 M4 M5 M6 M7
Peroxidase
(POX)
- + + + +/- - +/- -
Sudan Black
B
- + + + +/- - +/- -
Unspecific
esterases
- - - - + + - -
PAS - - - - - - + -

Cytochemical staining for myeloperoxidase is important in establishing
the lineage of myeloblasts

37. Immunophenotyping
FAB Immunological marker
AML with minimally differentiatedCD13,CD34, HLA-DR,
CD33,CD117,CD2,CD7,TdT
AML without maturation CD13,CD14,CD33, CD34
AML with maturation and with
t(8;21)
CD34,CD56
Acute promyelocytic leukemia CD13,CD33, HLA-DR absent, CD34
negative
Acute myelomonocytic leukemia
with abnormal eosinophils and
inversion 16
CD13,CD34,CD11b,CD11c,CD14,CD33
Acute monocytic leukemia and
11q23 abnormalties
CD14,CD4,CD36,CD64
Erythroleukemia Glycophorin 7, Transferrin receptor
CD71
Acute Megakaryocytic leukemia cCD41,cCD42b,cCD61

38. Minimally Differentiated Acute
Myeloid Leukemia
•5% of AML cases
•No definite evidence of myeloid differentiation can be given
by morphology & cytochemistry.
•CRITERIA FOR DIAGNOSIS
•<3% of blast which are MPO/SBB+(evident on EM)
•>20 % of leukemia cells expressing myeloid antigens.

39. Morphologically undifferentiated
blasts with distinct nucleoli are
peroxidase-negative and do not
show the esterase reaction typical of
monocytes
Bone marrow smear from the same
patient. Immunocytochemical detection
of CD13. A large proportion of the
blasts are positive (red).

40. AML without maturation
10 – 20% of AML cases
CRITERIA FOR DIAGNOSIS
Predominance of myeloblast ( > 90% ) without evidence of
maturation ( < 10% promyelocytes or others) in marrow .
IF no auer rods , at least 3% of blast must be MPO OR
SBB positive .
Median age : 45-50 yrs.
Generally chemosensitive and prognostically favourable
unless hyperleukocytosis or complex karyotype present.

41. BLOOD SMEAR BONE MARROW SMEAR
LARGE & SMALL BLAST WITH NORMAL
PLATELETS
BLAST WITH PALE TO BASOPHILIC AGRANULAR
CYTOPLASM , NUCLEI WITH FINE CHROMATIN &
PROMINENT NUCLEOLI

42. MYELOBLAST TYPES
TYPE 1
NO cytoplasmic granules
TYPE 2
15 – 20 CYTOPLASMIC
GRANULES

43. •TYPE 3
•>20 CYTOPLASMIC
GRANULES

44. AML with maturation and with t(8;21)
•30-45% of AML cases (Most frequent).
•Genes involved in t(8;21) are AML1 at 21q22 and ETO
(eight twenty one) at 8q22.
•CRITERIA FOR DIAGNOSIS
• Blast 20% or more(20-89) of all nucleated cells in bone
marrow
•Mature cells (promyelocytes to granulocytes) > 10%
•Monocytic cells < 20%.

45. BONE MARROW SMEAR
BLAST WITH NUCLEOLI AUER RODS

46. Acute promyelocytic leukemia
•Median age 30-38 yrs (young patient).
•It is generally not preceded by myelodysplastic
syndrome.
•Most patient present with hemorrhagic manifestation
secondary to DIC.
•Associated with t(15;17).
•Retinoic acid receptor (RAR- alpha) gene on
chromosome 17q12.
•Promyelocytic gene (PML gene) on chromosome 15q22.

47. AML and disseminated intravascular
coagulation (DIC).
Extensive purpura is present on the soles
of a patient with acute promyelocytic
leukemia and DIC

48. Acute promyelocytic leukemia
•Either HYPERGRANULAR OR MICROGRANULAR.
•Hypergranular type is most common.
•Leukopenia is seen in Hypergranular APL.
•Leukocytosis in Microgranular APL.

49. BONE MARROW BONE MARROW
SMEARSMEAR
HYPERGRANULAR
Nucleus : Folded, lobulated, granular obscure border.
Cytoplasm: Prominent Azurophilic granules.
Auer rods: Frequent, FAGGOT cells ( cells with bundles of auer rods)

50. Variations on the appearance of “faggot cells” in several different cases of APL.

51. BONE MARROW BIOPSY

52. MICROGRANULAR
Nucleus : Irregular, Folded. Mostly binucleated.
Cytoplasm : Fine small granules, “Dusky “ appearance.
Auer rods: Rare.

53. ACUTE MYELOMONOCYTIC LEUKEMIA with ABNORMAL
EOSINOPHILS and INVERSION Of Chrosome 16
•15-25% of AML cases
•CRITERIA FOR DIAGNOSIS
Blast >20%
Monocytic cells & their precursor
Neutrophil & their precursor
•Median age : 40 – 45 yrs.
•Leukocytosis is present in most of the patients.
•Prognosis is better than M1, M2, or M3.
>20%

54. Peripheral smear
myeloblast
MYELOCYTE
PROMONOCYTE

55. BONE MARROW SMEAR
MONOCYTES & NEUTROPHIL AT VARIOUS STAGES OF MATURATION

56. EOSINOPHIL
M4Eo
Immature eosinophils have a monocytoid nucleus and a mixture of eosinophilic
and large atypical basophilic granules. M4Eo(CBFb/MYH11)

57. Acute monocytic leukemia & 11q23
abnormalities
•Two types : M5a and M5b
•M5a :-
Poorly differentiated
Trisomy 8 is most common abnormality seen.
•M5b :-
Well differentiated.
FLT3 mutation is most common abnormality seen.
•Extramedullary disease occur in > 50% of the patient.
•It has a very poor prognosis , 6 -12 months

58. Leukemia cutis most commonly occurs in
monocytic forms of AML and represents
skin infiltration by leukemic blast cells
Gingival Hyperplasia

59. M5a( Acute Monoblastic Leukemia )
BLOOD SMEAR BONE MARROW SMEAR
MONOBLAST
80% or more are MONOBLAST
Abundant cytoplasm
Round nuclei with nucleoli
MONOBLAST WITH ABUNDANT CYTOPLASM
WITH FINE GRANULES

60. M5b( Monocytic Leukemia )
BLOOD SMEAR BONE MARROW SMEAR
PROMONOCYTES
<80% Monoblast
Mature monocytes or
promonocytes predominate

61. ACUTE ERYTHROID LEUKEMIA
M6a (ERYTHROLEUKEMIA)
5% of AML cases
More COMMON THAN pure erythroid leukemia.
Bimodal distribution- <20 yrs and >60yrs.
CRITERIA FOR DIAGNOSIS
>50% of nucleated marrow cells are erythroid lineage
>20% of nonerythroid cells are myeloblast
Dyserythropoiesis is prominent

62. BLOOD SMEAR BONE MARROW SMEAR
ERYTHROID PRECURSOR

63. M6b (PURE ERYTHROID LEUKEMIA )
Very rare
Also called ERYTHEMIC MYELOSIS ,
ACUTE Di GUGLIELMO SYNDROME
>80% of marrow cells are erythroblast
No significant myeloblastic component
ACUTE ERYTHROID LEUKEMIA

64. Bone marrow smear
Abnormal erythroid precursors

65. ACUTE MEGAKARYOBLASTIC
LEUKEMIA
•10% of AML in children & 5% of adult AML
•Bimodal distribution- Infancy and elderly
CRITERIA FOR DIAGNOSIS
•Megakaryoblast 20% or more in BM
•Bone marrow fibrosis
Megakaryoblast are either small to round with scanty
cytoplasm & coarse chromatin (resembling
lymphoblasts) or medium to large with fine chromatin &
prominent 1-3 nucleoli

66. ACUTE MEGAKARYOBLASTIC
LEUKEMIA
•Morphologically confused with
- L2 subtype of ALL
- AML M1.
•Diagnosis depends on expression of at least one platelet
antigen ( i.e., CD41,CD42b, CD61 or factor VIII related
antigen)
•Most common leukemia seen in Down’s Syndrome.
•Platelet show impaired aggregation response.
•Elevated serum Lactate Dehydrogenase level.

67. •Blast show distinct cytoplasmic blebs or psedopods
formation
•Peripheral blood – fragments of megakaryoblast
micromegakaryocytes Or dysplastic large platelets seen

68. Promegakarocytes , irregular nuclei , coarse chromatin
BONE MARROW SMEAR

69. BONE MARROW BIOPSY
PROLIFERATION OF MEGAKARYOCYTES

70. AML WITH MULTILINEAGE
DYSPLASIA
•Multilineage dysplasia – dysplasia present
>50% of cells in 2 or more myeloid cell lines
•Occur in elderly
•With / without prior h/o MDS
•Poor prognosis
•Chromosomal abnormalities similar to MDS

71. An aspirate smear with increased
blasts and a dysplastic mature
erythroid precursor displays
irregular (“cookie cutter”) nuclear
contours
(arrow).

72. This aspirate smear shows several
giant hypogranular bands (arrows)
and a dysplastic erythroid
precursor with asymmetric bi-
nucleation is situated just below the
centrally located hypogranular band.

73. An aspirate smear reveals increased
blasts and two dysplastic
micromegakaryocytes (arrows).

74. AML & MDS therapy related
•Different from denovo AML
•Characteristic cytogenetic abnormalities
•Multilineage dysplasia
•Refractoriness to therapy
•Short survival
•Follow TOPOISOMERASE II INHIBITOR
( myeloid/lymphoid) OR
ALKYLATING AGENTS

75. IMMATURE BASOPHIL PRECURSOR
Basophilic differentiation
Blast contain basophilic granules
ACUTE BASOPHILIC LEUKEMIA

76. Positive metachromatic staining with TOLUIDINE BLUE

77. ACUTE PANMYELOSIS WITH
MYELOFIBROSIS
•Very rare type.
•Median age – 57 to 67 yrs.
•Pancytopenia with < 5% blast.
•No history of preceding myeloproliferative disorder.
•Proliferation of all major myeloid cell lines
•Dyspalstic changes are present along with fibrosis of bone
marrow

78. Relacement by blast & fibrous tissue
Bone marrow biopsy

79. MYELOID (granulocytic) SARCOMA
(Myeloblastoma)
•Isolated tumour mass.
•Also known as Chloroma because some appear green or
turn green in dilute acid secondary to expression of MPO.
•Composed of myeloblast or immature cells in
extramedullary site
•Sign of relapse in a treated case of AML
•Common sites – orbits and the paranasal sinuses.
•The diagnosis should be suspected if eosinophilic
myelocytes are present in H & E stained biopsy sections.

80. Differential diagnosis
•1. Leukaemoid reaction
•2. Myelodysplastic Syndrome
•3. Acute Lymphoblastic Leukemia
•4. Blast crisis of Chronic Myeloid
Leukemia

81. LEUKAEMOID REACTION
•Refers to the presence of markedly increased leucocyte
count (>50,000/mm3) and immature white blood cells in
peripheral blood resembling leukemia but occurring in
non-leukaemic conditions.
•Causes of leukaemoid reaction-
Severe bacterial or viral infection.
Severe acute haemolysis.
Severe haemorrhage
Cancer metastatic to bone marrow.
Tuberculosis

82. LEUKAEMOID REACTION
Differentiation from AML is made by following features:
•Clinical presentation.
•Presence of underlying disease.
•Morphology on blood smear.
•% of blasts in bone marrow.
•Correction of leukaemoid blood picture after treatment of
underlying disease.

83. Myelodysplastic syndrome
•Differentiation of AML from MDS depends on proportion
of myeloblasts in the bone marrow.
•In AML, myeloblasts are greater than 20%.
•In MDS, myeloblasts are less than 20%.
•MPO staining may also be useful for diagnosis of MDS
wherein granulocytes may lose MPO reactivity.

84. ALL Vs AML
ALL AML
Age Mainly children Mainly adults
Lymphadenopathy Usually present Usually absent
Gum hypertrophy -ve +ve in M4/M5
Skin infiltration -ve +ve in M4/M5
Granulocytic sarcoma-ve +ve in few cases
Mediastinal mass +ve in T-ALL -
Associated DIC -ve +ve in M3

85. Blast crisis of CML
•Presence of marked splenomegaly, basophilia and
Philadelphia chromosome are suggestive of CML .
These features differentiate blast crisis of CML with
AML.

86. Prognosis
Factors Favourble Unfavourable
Clinical factors
Age < 45 yrs < 2yrs , > 60 yrs
Leukemia De novo -Antecedent
hematological disorder
-Myelodysplastic disorder
-- Myeloproliferative
disorder
Infection Absent Present
Prior chemotherapy No Yes
Leukocytosis < 25,000 / mm3 > 100,000 /mm3
Serum LDH Normal Elevated
Extramedullary diseaseAbsent Present
CNS disease Absent present

87. Prognosis
Factors Favourble Unfavourable
Morphology
Auer rods Present Absent
Eosinophils Present Absent
Megaloblastic erythroidAbsent Present
Dysplastic
megakaryocytes
Absent Present
FAB type M2,M3,M4 M0,M6,M7

88. Prognosis
Factors Favourble Unfavourable
Surface / Enzyme
Marker
Myeloid CD34 –ve , CD 14 –ve,
CD 13 -ve
CD 34 +ve
HLA- DR Negative Positive
TdT Absent Present
Lymphoid CD 2 +ve CD 7 +ve , CD 56 +ve,
Biphenotypic
Multidrug resistance
gene
Absent Present
Cytogenetics t (15;17), t (8;21),
inv (16)
Monosomy 7, del (7q)
Monosomy 5, del (5q),
Complex karyotype

89. THANK YOU
Presented by Dr. Monika Nema