Leukemias-basic pathology

LEUKEMIAS
Guided by:
Dr.Ami shah
Moderated by:
Dr.Priyanka gohel
Presented by:
Jaimin Manek

 Leukemia: is characterized by abnormal
proliferation of blood cells,usually
WBCs(Leukocytes).
 Acute leukemia: rapid increase of immature
blood cells.
 Chronic leukemia: excessive build up of
relatively mature, but still abnormal blood cells.

 Leukemia results
- From somatic mutations in the DNA.
- By activating oncogenes or deactivating
tumor suppressor genes.
FACTORS:
 Ionizing radiation
 Viruses: HumanT-lymphotropic virus (HTLV-1)
 Chemicals: Benzene,chemotherapy.
 Smoking: slight increase in leukemia incidence.
 Genetic predisposition: Down syn.,Fanconi anemia

Normal hemopoiesis is finely tuned by hemostatic
feedback mechanisms involving cytokines and
growth factors that modulate the marrow output of
red cells, granulocytes and platelets.
These mechanisms are deranged in marrows involved
by myeloid neoplasms.
Loss of control on growth & survival and suppressor
fuctions.
CSBRP-SDUMC-Oct-2014

Main groups:
1-Myeloid (acute / chronic)
2-Lymphoid (acute / chronic)
3-Mixed lineage leukemias

 The most common malignant
neoplasms of childhood are Leukemias
 Which one is the commonest type of
childhood Leukemias?
AML 10%
ALL 80%
CML 2-3%
JCML 1-2%

Myeloblast Promyelocyte Myelocyte Metamyelocyte Band Neutrophil
MATURATION
Adapted and modified from U Va website
Myeloid maturation:

Two types of classifications:
1- FAB classification
- degree of maturation & lineage of blasts
- usage of cytochemistry & IHC
2-WHO classification
- degree of maturation & lineage of blasts
- Immunophenotyping
- cytogenetic & molecular features
- clinical outcome

1. M0: minimally differentiated
2. M1: myeloblastic leukemia without maturation
3. M2: myeloblastic leukemia with maturation
4. M3: hypergranular promyelocytic leukemia
5. M4: myelomonocytic leukemia
6. M4Eo: variant, increase in marrow eosinophils
7. M5: monocytic leukemia
8. M6: erythroleukemia (DiGuglielmo's disease)
9. M7: megakaryoblastic leukemia

1. AML with recurrent genetic abnormalities
2. AML and MDS
3. AML -Tx related
4. AML not otherwise categorised
5. Myeloid Sarcoma
6. Myeloid proliferation related to Down
Syndrome

 Most of the signs and symptoms are due to:
1-Anemia.
2-Leukopenia.
3-Thrombocytopenia.
 Bicytopenia, Pancytopenia.
 All symptoms associated with leukemia
can be attributed to other diseases,
consequently, leukemia is always
diagnosed by laboratory investigations.

 AML M0, M1, M2 : Chloromas
 AML M3 : DIC
 AML M4, M5 : Gum hypertrophy
 AML M7 : Mediastinal mass
(germ cell tumors)

1. CBC a. Anemia
b. Trombocytopenia
c. WBC
High Normal Low
2. Coagulation Studies (M3-DIC)
3. Biochemical Studies

Bone marrow (>20% blasts)
- Morphology
- Cytochemistry
- Immunophenotyping
- Cytogenetics
Peripheral blood examination
 blasts in almost all cases
Molecular Genetic Analysis

The diagnostic requisite of 20 percent type I and II
myeloblasts in the peripheral blood or bone marrow.
BLAST Equivalents:
1. In AML (M3), the predominant leukemic cell is
promyelocyte
2. In AML (M5A), the predominant proliferating cell is the
monoblast
3. In AML (M5B), the predominant cell is the
promonocyte.
4. The megakaryoblasts of acute megakaryoblastic
leukemia vary in morphology but uniformly lack the
cytochemical properties of myeloblasts.CSBRP-SDUMC-Oct-2014

 < 1% of the normal bone marrow, not observed in
normal blood
 Vary in size, but are usually large
 Nucleus is delicate, large, round or oval, with
prominent nucleoli. Stain purplish red with
Wright stain. Chromatin stains evenly
 Small to moderate amount of bluish nongranular
cytoplasm
 Three major types:Type I, II, and III

 Fine nuclear chromatin
 2 to 4 distinct nucleoli
 Moderate rim of pale to basophilic
cytoplasm
 Without azurophilic granules
Type II
Delicate azurophilic granules in
the cytoplasm (up to 20)
Type III
Numerous azurophilic granules
in the cytoplasm

Monoblast Myeloblast

 Erythroblast
Megakaryoblast Lymphoblast

CYTOLOGIC FEATURES OF BLASTS IN AML & ALL
AML ALL
Blast size Medium to large, uniform Variable Small to medium
Cytoplasm
Fine granules may be
present
Usually scant, a few coarse
granules may be seen
Auer rods
Present in 60-70% of
cases
absent
Nuclear
chromatin
Finely dispersed Fine to coarse
Nucleoli 2-4, prominent 1-3, indistinct

Is an azurophilic linear structure (Single or multiple)
present in numerous blasts or in rare cells
• Present approximately 60 to 70 % of cases of AML.
• MPO, SBB, and CAE positive
The presence of an Auer rod in one or more blasts is
definitive evidence of AML.
(The finding is not specific for any one type of AML.)

CYTOCHEMICAL PROFILES OF ACUTE LEUKEMIAS
MPO
/ SBB
CAE NSE PAS AP
ALL _ _ + / -
Focally
+
75%
+ / -
Focal in T-
ALL
AML + +
+
Monocytic-
diffuse
- / + +
MPO-myeloperoxidase, SBB-Sudan balck B, CAE-chloracetate esterase, NSE-
non specific esterase, PAS-periodic acid schiff, AP-acid phosphataseCSBRP-SDUMC-Oct-2014
cvcv

Myeloperoxidase
(MPO)
p-Phenylene diamine +Catecol + H2O2
MPO > Brown black deposits

 It is a direct stain phospholipid in granular membrane.
 Auer rods are MPO and SBB positive.

Chloracetate (Specific)
Esterase
Myeloid Cell Line
Naphthol-ASD-chloracetate
Free naphthol compounds
+
Stable diazonium salt
Red deposit

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
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

Chromosomal
Abnormality
Leukemia
t(8;21) M2
t(15;17) M3
inv, del, t(16q) M4
t(9;11) M5 (M5a); M4

 5% of AML cases
 No definite evidence of myeloid differentiation can be given by
morphology & cytochemistry.
 Blasts resembles M1 blast and L2 Lymphoblast
PS: Large cells with pale grey blue CYTOPLASM
NUCLEUS has opened fine chromatin with >= 1 nucleoli
Cytochemistry:
< 3% blasts reactive for MPO, SBB or NSE
Immunophenotyping:
 20% blasts express one or more myeloid antigens: CD13,
CD14, CD33
 may beTdT positive;

AML-M0 Bone marrow smear, May-Giemsa stain, x1000

AML M0 Bone marrow smear, cyMPO stain, x1000

15-20% of all AML cases
M1 is differentiated from M2 by the fact that
>90% blasts of non erythroid cells
<10% of marrow nucleated cells are promyelocytes or more
mature neutrophils
PS: Blasts have variable N/C ratio with pale basophilic CYTOPLASM
NUCLEUS has 1-4 nucleoli
Cytochemistry:>3% blasts reactive for MPO or SBB.
Immunophenotyping: Blasts express myeloid antigens: CD13, CD14,
CD33.

BLOOD SMEAR BONE MARROW SMEAR
BLAST WITH PALE TO BASOPHILIC AGRANULAR CYTOPLASM ,
NUCLEI WITH FINE CHROMATIN & PROMINENT NUCLEOLI

Commonest (30%) of all AML
Evidence of maturation to promyelocytes and more mature
neutrophils in 10 percent or more of the cells.
PS: Blasts are large with pale basophilic CYTOPLASM
NUCLEUS: oval to indented with 2-3 nucleoli
Cytochemistry: MPO, SBB, CEA +ve
Immunophenotyping: CD15, CD13, CD33 +ve
In t(8,21) associated cells: Pathognomonic
40-80% are positive for CD19
20% areTdT positive

AML M2 CASE 1 Bone marrow smear, MGG x200

A form of AML characterized primarily by a
proliferation of abnormal promyelocytes.
It is usually accompanied by
• DIC
• t(15;17)
The disease presents in two morphologic types:
1) hypergranular APL
predominant cell is an abnormal promyelocyte with
markedly increased and coarse azurophilic granules
2) microgranular or hypogranular APL
predominant cell is an abnormal promyelocyte with
diminished or small azurophilic granules.

The most common presenting symptoms, occurring in
90 % of patients, relate to hemorrhagic manifestations
and include
easy bruisability,
bleeding gums,
hemoptysis,
epistaxis, petechiae,
symptoms of gastrointestinal &
intracranial hemorrhage.
Basic pathology is DIC.

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

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

ACUTE MYELOMONOCYTIC LEUKEMIA
 Nearly 20% of all AML cases
 Both MYELOBLAST and MONOBLAST co exist
 M.c. Acute Leukemia in INFANTS
P.S : Both Myeloblast and monocytic components
( monoblasts, promonocytes, monocytes ) seen
Cytochemistry : monocytic cells – NSE stain
myeloid cells –Mpo/CAE stain
IFT : Monocytic component- CD64,CD14
Myeloid component- CD13,CD33,CD15

Peripheral smear
MYELOBLAST
MYELOCYTE
PROMONOCYTE
Large monocytoid cells with
Cytoplasm: Pale blue agranular
Nuclei: Ovoid to reniform

>80% of leukemic cells are monocytic lineage
• Neutrophil component may constitute <20%
• Acute monoblastic M5a Vs monocytic leukemias M5b:
PS: Monoblasts >80% in monoblastic leukemia
Promonocytes are predominant in monocytic leukemias
Cytochemistry :SBB – Fine scattered granules in monoblast
NSE – Diagnostic
IFT: CD14, CD11b, CD11c, CD64, CD68

MONOBLAST
 80% or more are MONOBLAST
 Abundant cytoplasm
 Round nuclei with nucleoli
MONOBLAST WITH ABUNDANT
CYTOPLASM WITH FINE GRANULES

BLOOD SMEAR
BONE MARROW SMEAR
PROMONOCYTES
 <80% Monoblast
 Mature monocytes or
promonocytes predominate

 Def: Erythroleukemia by definition involves both the
granulocytes and erythroid cells
TYPES:
M6a Erythroleukemia
M6b PURE erythroleukemia
Erythroblast:
 Relatively high nuclear/cytoplasmic ratio
 Nucleus round with slightly condensed chromatin;
 Nucleoli variably prominent
 Deeply basophilic cytoplasm that may be vacuolated

 5% of AML cases
 More COMMONTHAN 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
Cytochemistry:
MPO +ve, PAS +ve
IFT: Glycophorin A +, CD13, CD33, CD117

ERYTHROID PRECURSOR

 Very rare
 Also called ERYTHEMIC MYELOSIS ,
ACUTE Di GUGLIELMO SYNDROME
 >80% of marrow cells are erythroblast
 No significant myeloblastic component

Bone marrow smear
Abnormal erythroid precursors

AML M6 Case-3 Bone marrow smear, May-Giemsa stain, x1000

History: 47year old man with a
history of renal transplant as
well as refractory anemia with
ringed sideroblasts diagnosed 1
year back.
Now he has fatigue and loss of
weight. He is on cyclosporin
and prednisolone.
Source:CAP
AML M6b case-5

Investigations:
Blood count revealed anemia and thrombocytopenia and rare
blasts.
BM revealed 81% erythroid precursors, marked dysplastic
changes and “block-and-blush” PAS positivity of erythroid
lineage. There were 12% myeloblasts and minimal dysplasia of
granulocytic cell line.
Diagnosis:
Acute erythroleukemia-M6b (pure erythroleukemia).
AML M6b case-5
Source:CAP

 10% ofAML in children & 5% of adult AML
 Bimodal distribution- Infancy and elderly
 Most common leukemia seen in Down’s Syndrome.
 May be associated with mediastinal germ cell tumors
CRITERIA FOR DIAGNOSIS
 Megakaryoblast 20% or more in BM
 Bone marrow fibrosis
MK blast:
 12-18µm
 Round nucleus with reticular
chromatin
 1-3 nucleoli
 Cytoplasm is basophilic and agranular
 Cytoplasmic blebs
 May resemble Lymphoblast

 Morphologically confused with
- L2 subtype ofALL
- AML M1.
Diagnosis depends on
Elevated serum Lactate Dehydrogenase level.
Marked leucocytosis
Cytochemistry : MPO, SBB,TdT are negative
Some scattered PAS positivity
Immunophenotyping : CD41, CD61, Gp IIb/IIIa

 Blast show distinct cytoplasmic blebs or psedopods formation
 Peripheral blood – fragments of megakaryoblast
micromegakaryocytesOr dysplastic large platelets seen

Promegakarocytes , irregular nuclei , coarse chromatin
BONE MARROW SMEAR

 Favorable:
 younger age (<50)
 WBC <30,000
 t(8;21) – seen in >50% with AML M2
 inv(16) – seen in AML M4 eos
 t(15;17) – seen in >80% AML M3
 Unfavorable:
 older age (>60)
 Poor performance status
 WBC >100,000
 Elevated LDH
 prior MDS or hematogic malignancy
 CD34 positive phenotype, MRD1 postive phenotype
 del (5), del (7)
 trisomy 8
 t(6;9), t(9;22)
 t(9;11) – seen in AML M5
 FLT3 gene mutation (seen in 30% of patients)

 CML is a clonal stem cell disorder characterised
by increased proliferation of myeloid elements at
all stages of differentiation.
 15% of leukaemias.
 It occurs most often between 40–60yrs
 Incidence increases with age, M > F.

Philadelphia chromosome
 Present in >80% of those with CML. It is a
hybrid chromosome comprising reciprocal
translocation between the long arm of
chromosome 9 and the long arm of
chromosome 22—t(9;22) forming a fusion gene
BCR/ABLon chromosome 22, which has
tyrosine kinase activity

CML is characterised by 3 distinct phases
Chronic Phase:
Proliferation of myeloid cells, which show a full range
of maturation.
Accelerated Phase: decrease in myeloid differentiation
occurs.
Blast crisis: (acute leukemia)

1) Chronic granulocytic leukemia
Classical CML with Ph chromosome +ve/ -ve
But bcr/abl 1 +ve
2) Atypical CML or Ph –ve CML
Monocytosis is intermediate b/w Classical CML & CMML
( 3-10%)
bcr/abl 1 –ve
3) Chronic myelomonocytic leukemia (CMML)
Ph –ve CML with predominance of monocytes in blood
( >10%)

Symptoms
 Asymptomatic (50% of patients)
 Fatigue
 Weight loss
 Abdominal fullness and anorexia
 Abdominal pain, esp splenic area
 Increased sweating
 Easy bruising or bleeding
Signs
 Splenomegaly (95%)
(50% of patients have a palpable spleen ≥ 10 cm BCM, Usually firm and non
tender)
 Hepatomegaly (50%)

Approximately 85%of patients are diagnosed in the chronic
phase and then progress to the accelerated and blast phases
after 3-5 years.
The diagnosis of CML is based on
Histopathologic findings in the peripheral blood
&
Philadelphia chromosome in bone marrow cells
Investigation
 CBC with differential
 peripheral blood smear
 bone marrow analysis
 US using for liver/spleen

Peripheral blood – neutrophils 20,000 - >500, 000/ L
- basphilia
-  LAP score
- blasts < 5%
- Nucleated RBCs
- Thrombocytosis
- Anaemia
 Neutrophils and Myelocytes are PREDOMINANT
cells with blasts being < 3%.

 PS shows increased Myelocytes and Mature
Polymorphs

Leukemoid
reaction
CML
WBC High High
Anemia (-) (+)
PBS Shift to the Left
Toxic granulation
Dohle bodies
Shift to the left (blast)
Eosinophilia,
basophilia
LAP score High Low
Philadelphia
chromosome
(-) (+)

 Leukemias are clonal disorders
 Mutations in oncogenes is the most common
underlying pathology
 Present with: Anemia, Petichiae, infections,
hepatosplenomegaly, Lymphadenopathy
 There may be normal, low or elevated total white
count.

 AML is a heterogeneous disease
 Blast count should be 20% in BM
 There are blast equivalents
 The presence of an Auer rod is definitive
evidence of AML
 WHO classification is well accepted
 Detection of genetic abnormalities dictates
Tx and Prognosis

Leukemias-basic pathology

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