Week 10: Leukemia

 AML  ORO
 ALL  CD markers
 FAB classification  Flowcytometry
 WHO classification  CML
 Cytochemistries  CLL
 MPO  Karyotype
 SBB  Ph chromosome
 PAS  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
 Typical Labs
of AML
 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
FAB (1976) Classification
 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

M4 M3

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
 FAB criteria  WHO criteria
 Morphology
 Morphology
 Immunophenotyping
 Cytochemistry
 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
 AML with Recurrent Cytogenetic
Translocations (WHO 1995)
 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
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)
 WHO Classification of
Lymphoproliferative Syndromes

 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 •+ •0 to +
•Tdt •+ •+
•CD34 •+ •0

•CD19 •+ •Pre-T
•C 22 •cALL and older •Pre-T
•CD10 •cALL and older •Pre-T
•CD20 •Pre-B and older •0
•Cyt- •Pre-B and older •0
•SIg •B-ALL •0

•CD7 •0 •+
•CD3 •0 •+
•CD5 •0 •+
•CD2 •0 •T-ALL
•CD1 •0 •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 Hypoploidy
Trisomy 4, 10, 17 Trisomy 5
t(12;21) (TEL/AML1) t(1;19 (E2A/PBX1)
Mixed lineage leukemia
T(9;22) (Ph)

BM blast count Mkd reduction at day 7 Mild reduction at day 7

during induction
Typical Labs in CML
 Leukocytosis with blastemia
 Thrombocytosis
 Basophilia
 Micro-megakaryocytes
 Low LAP score (intermediate if infected)
 About 10% blasts in BM
 Philadelphia chromosome
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
 0 1+

2+ 3+ 4+
LAP Score
Example:

0 x 35 cells = 0
1+ x 30 cells = 30
2+ x 20 cells = 40
3+ x 10 cells = 30
4+ x 5 cells = 20
120 LAP Score
Philadelphia Chromosome
 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
 WHO Classification of MPS
(1997)
 CML becomes
 CML, Ph + t(9;22) BCR/ABL

 Chronic Neutrophilic Leukemia (CNL)

 Chronic Eosinophilic Leukemia and Hyper-

eosinophilic Syndrome (CEL/HES)
 PV remains PV
 ET remains ET
 AMM becomes
 Chronic Idiopathic Myelofibrosis
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
 CLL
PB and BM
Bone Marrow
WHO Lymphoid Neoplasms
 B cell neoplasms
 T/NK cell neoplasms
 Hodgkin lymphoma (disease)
Mature B Cell Neoplasms
 B cell CLL/SLL  Hairy cell leukemia
 B prolymphocytic  Lymphoplasmocytic
leukemia leukemia
 Burkitt’s lymphoma /  Mantle cell lymphoma
leukemia  Plasma cell myeloma /
 Splenic marginal zone plasmacytoma
B lymphoma  Follicular lymphoma
 Extranodal marginal B  Diffuse large B
lymphoma lymphoma
T/NK Cell Neoplasms
 T prolymphocytic  Mycosis fungoides
leukemia (Sezary syndrome)
 Anaplastic large cell
 T granular
lymphoma
lymphocytic leukemia
 Hepatosplenic T
 Aggressive NK cell lymphoma
leukemia  Peripheral T lymphoma
 Adult T lymphoma /  Immunoblastic T
leukemia lymphoma