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.
12. Differences between FAB and WHO
FAB-classification:
1) Heavily used âMorphologic Findingsâ
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.
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 âblastâ
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. L y Mmoprhpohbolloagsty Myeloblast
Nuclear
chromatin
Coarse Fine
Nucleoli 1-2 3-5
N:C ratio High High
Auer rod Absent Present
Accompanying
Lymphocytes Myeloid
cells
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.
â 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 (âĽ50%) 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.
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 â naphthyl
acetate.
35. PAS staining
⢠Periodic Acid Schiff stain reacts primarily with glycogen,
generating a fuchsian coloured precipitate.
PAS+ MEGAKARYOCYTES
36. AML â 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 differentiated CD13,CD34, HLA-DR,
CD33,CD117,CD2,CD7,TdT
AML without maturation CD13,CD14,CD33, CD34
AML with maturation and with
CD34,CD56
t(8;21)
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
ď§ 10 â 20% of AML cases
ď§ 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 .
ď§ Median age : 45-50 yrs.
ď§ 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 â 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%.
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.
52. MICROGRANULAR
Nucleus : Irregular, Folded. Mostly binucleated.
Cytoplasm : Fine small granules, âDusky â 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
>20%
⢠Median age : 40 â 45 yrs.
⢠Leukocytosis is present in most of the patients.
⢠Prognosis is better than M1, M2, or M3.
55. BONE MARROW SMEAR
MONOCYTES & NEUTROPHIL AT VARIOUS STAGES OF MATURATION
56. M4Eo
EOSINOPHIL
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
ď80% or more are MONOBLAST
ďAbundant cytoplasm
ďRound nuclei with nucleoli
MONOBLAST WITH ABUNDANT CYTOPLASM
WITH FINE GRANULES
60. M5b( Monocytic Leukemia )
BLOOD SMEAR BONE MARROW SMEAR
PROMONOCYTES
ď<80% Monoblast
ďMature monocytes or
promonocytes predominate
61. ACUTE ERYTHROID LEUKEMIA
M6a (ERYTHROLEUKEMIA)
ď5% of AML cases
ďMore COMMON THAN pure erythroid leukemia.
ďBimodal distribution- <20 yrs and >60yrs.
CRITERIA FOR DIAGNOSIS
ď>50% of nucleated marrow cells are erythroid lineage
ď>20% of nonerythroid cells are myeloblast
ďDyserythropoiesis is prominent
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 Downâs Syndrome.
⢠Platelet show impaired aggregation response.
⢠Elevated serum Lactate Dehydrogenase level.
67. ⢠Blast show distinct cytoplasmic blebs or psedopods
formation
⢠Peripheral blood â fragments of megakaryoblast
micromegakaryocytes Or dysplastic large platelets seen
70. AML WITH MULTILINEAGE
DYSPLASIA
⢠Multilineage dysplasia â 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 (âcookie cutterâ) 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
77. ACUTE PANMYELOSIS WITH
MYELOFIBROSIS
⢠Very rare type.
⢠Median age â 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
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 â orbits and the paranasal sinuses.
⢠The diagnosis should be suspected if eosinophilic
myelocytes are present in H & E stained biopsy sections.
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-
ď Severe bacterial or viral infection.
ď Severe acute haemolysis.
ď Severe haemorrhage
ď Cancer metastatic to bone marrow.
ď Tuberculosis
82. LEUKAEMOID REACTION
ďź 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.