Week 10: Leukemia
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AML
ALL
FAB classification
WHO classification
Cytochemistries
MPO
SBB
PAS
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ORO
CD markers
Flowcytometry
CML
CLL
Karyotype
Ph chromosome
LAP
Signs and Symptoms of AML
Insidious nonspecific onset
 Pallor due to anemia
 Febrile due to ineffective WBC
 Petechiae due to thrombocytopenia
 Mucus membrane and gum bleed in
M4 and M5
 Bone pain
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Typical Labs
of AML
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Leukocytosis
Blastemia
Leukemic hiatus
Auer rods in M2, M3,
M4
Thrombocytopenia
Anemia
>20% blasts in BM
Other Findings
CD 13 and CD 33 in flowcytometry
 Cytochemistries
 Myeloperoxidase
 Sudan black B
 Choloroacetate esterase (specific)
 Nonspecific esterase
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FAB (1976) Classification
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M0 -- Undifferentiated AML
M1 -- AML without maturation
M2 -- AML with maturation
M3 -- Acute Promyelocytic Leukemia
M4 -- Acute Meylomonocytic Leukemia
M5 -- Acute Monocytic Leukemia
M6 -- Erythroleukemia (DiGuglielmo’s)
M7 -- Megakaryoblastic Leukemia
M1 and M2
Myeloperoxidase
(MPO)
p-Phenylene diamine + Catecol + H2O2
MPO
> Brown black deposits
M3
M4
M5
Chloracetate (Specific) Esterase
Myeloid Cell Line
Naphthol-ASD-chloracetate
CAE > Free naphthol compounds
+ Stable diazonium salt (eg, Fast Corinth)
> Red deposit
Non-Specific Esterase
Monocytic Line
 Naphthyl acetate
ANAE > Free naphthyl compounds
+Stable diazonium salt (eg, Fast blue RR)
> Brown deposits
NSE with Fl inhibition
Histiocyte
Double Esterase in M4
FAB vs WHO Classifications of
Hematologic Neoplasm
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FAB criteria
 Morphology
 Cytochemistry
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WHO criteria
 Morphology
 Immunophenotyping
 Genetic features
 Karyotyping
 Molecular testing
 Clinical features
WHO Classification of AML
AML with recurrent cytogenic
translocations
 AML with multi-lineage dysplasia
 AML and myelodysplasia, therapy related
 AML, not otherwise categorized
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AML with Recurrent Cytogenetic
Translocations (WHO 1995)
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t(8;21) -- some maturation of neutrophilic line;
rare in older patients; AML1/ETO fusion protein;
>90% FAB M2
t(15;17) -- APL (granular and microgranular
variants); retinoic acid receptor (RAR) leukemias;
middle aged adults; DIC
inv(16) or t(16;16) -- monocytic and granulocytic;
abnormal eosinophilic component
11q23 -- monocytic; children; most common is
t(9;11)
FAB Classification of ALL
L1: Small homogeneous blasts; mostly in
children
 L2: Large heterogeneous blasts; mostly in
adults
 L3: “Burkitt” large basophilic B-cell blasts
with vacuoles
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L2
L3
Periodic Acid Schiff
Periodic acid + Glycogen
oxidation > Aldehyde + Schiff reagent
(para-rosaniline, Na metabisulfite)
> Red deposit
ALL Cytochemistries
Oil Red O: stains L3 vacuoles
 Terminal deoxynucleotidyl transferase
(Tdt): DNA polymerase in early
lymphoblasts
 Cell surface markers (CD’s)
 Cytoplasmic and surface immunoglobulins:
B-cell line
 T-cell receptor (TCR)
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WHO Classification of
Lymphoproliferative Syndromes
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Precursor B Lymphoblastic Leukemia/Lymphoma
(ALL/LBL) -- ALL in children (80-85% of
childhood ALL); LBL in young adults and rare;
FAB L1 or L2 blast morphology
Precursor T ALL/LBL -- 15% of childhood ALL
and 25% of adult ALL
Burkitt Leukemia/Lymphoma (FAB L3)
•Antigens
•B-Lineage
•T-Lineage
•HLA Dr
•Tdt
•CD34
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•+
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•0 to +
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•0
•CD19
•C 22
•CD10
•CD20
•Cyt-
•SIg
•+
•cALL and older
•cALL and older
•Pre-B and older
•Pre-B and older
•B-ALL
•Pre-T
•Pre-T
•Pre-T
•0
•0
•0
•CD7
•CD3
•CD5
•CD2
•CD1
•0
•0
•0
•0
•0
•+
•+
•+
•T-ALL
•T-ALL
Prognosis
Indicators
Favorable
Poor
WBC
< 50,000/L
 50,000/L
Age
1 - 10
< 1 or 10
Gender
Female
Male
Blast
B-cell
T-cell and mixed
Karyotype
Hyperploidy
Trisomy 4, 10, 17
t(12;21) (TEL/AML1)
Hypoploidy
Trisomy 5
t(1;19 (E2A/PBX1)
Mixed lineage leukemia
T(9;22) (Ph)
BM blast count Mkd reduction at day 7
during induction
Mild reduction at day 7
Typical Labs in CML
Leukocytosis with blastemia
 Thrombocytosis
 Basophilia
 Micro-megakaryocytes
 Low LAP score (intermediate if infected)
 About 10% blasts in BM
 Philadelphia chromosome
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CML
Bone marrow aspirate
and biopsy
Pseudo-Gaucher’s
cells in BM
Leukocyte Alkaline
Phosphatase (LAP)
Naphthol AS-MX phosphate LAP at pH8.6 >
Naphthol AS-MX + Diazonium salt
(eg, Fast blue RR)
> Insoluble pigment
LAP Score
Count 100 consecutive segs and bands
 Score:
0 = no granules
1+ = occasional diffuse granules
2+ = moderate number of granules
3+ = many strongly positive granules
4+ = confluent strongly positive granules
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0
2+
1+
3+
4+
LAP Score
Example:
0
1+
2+
3+
4+
x
x
x
x
x
35 cells
30 cells
20 cells
10 cells
5 cells
= 0
= 30
= 40
= 30
= 20
120 LAP Score
Philadelphia Chromosome
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9 and 22 translocation almost specific to CML
Karyotype to visualize Ph chromosome
Produces BCR/c-abl fusion oncogene
Gene product p190 is a hyperactive tyrosine
kinase
Ph chromosome seen in ALL produces p210 and
chronic neutrophilic leukemia produces p230
Karyotype 46,XX,t(9;22)(q34;q11.2) -- Ph chromosome
FISH showing the BCR (green), ABL (orange), and BCR-ABL fusion
signals (arrow): A=positive (contains a residual ABL signal), B=normal
FAB (1982) Classification of
Myeloproliferative Disease (MPD)
Chronic Myelocytic Leukemia (CML)
 Polycythemia Vera (PV)
 Essential Thrombocythemia (ET)
 Agnogenic Myeloid Metaplasia with or without
Myelofibrosis (AMM)
 Benign Leukemoid Reaction
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WHO Classification of MPS
(1997)
CML becomes
 CML, Ph + t(9;22) BCR/ABL
 Chronic Neutrophilic Leukemia (CNL)
 Chronic Eosinophilic Leukemia and Hypereosinophilic Syndrome (CEL/HES)
 PV remains PV
 ET remains ET
 AMM becomes
 Chronic Idiopathic Myelofibrosis
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Myelofibrosis
Myelofibrosis
Chronic Lymphocytic Leukemia
Exclusive in elderly
 Lyphocytosis unrelated to viral infection
 Hyper-mature lymphocytes with highly
condensed nuclei
 Smudge cells: preventable with a drop of
bovine albumin
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CLL
PB and BM
Bone Marrow
WHO Lymphoid Neoplasms
B cell neoplasms
 T/NK cell neoplasms
 Hodgkin lymphoma (disease)
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Mature B Cell Neoplasms
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B cell CLL/SLL
B prolymphocytic
leukemia
Burkitt’s lymphoma /
leukemia
Splenic marginal zone
B lymphoma
Extranodal marginal B
lymphoma
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Hairy cell leukemia
Lymphoplasmocytic
leukemia
Mantle cell lymphoma
Plasma cell myeloma /
plasmacytoma
Follicular lymphoma
Diffuse large B
lymphoma
T/NK Cell Neoplasms
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T prolymphocytic
leukemia
T granular
lymphocytic leukemia
Aggressive NK cell
leukemia
Adult T lymphoma /
leukemia
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Mycosis fungoides
(Sezary syndrome)
Anaplastic large cell
lymphoma
Hepatosplenic T
lymphoma
Peripheral T lymphoma
Immunoblastic T
lymphoma