1. Week 10: Leukemia AML ALL FAB classification WHO classification
Cytochemistries
MPO
SBB
PAS
ORO
CD markers
Flowcytometry
CML
CLL
Karyotype
Ph chromosome
LAP

2. 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

3. Typical Labs of AML Leukocytosis Blastemia Leukemic hiatus
Auer rods in M2, M3, M4
Thrombocytopenia
Anemia
>20% blasts in BM

4. Other Findings CD 13 and CD 33 in flowcytometry Cytochemistries
Myeloperoxidase
Sudan black B
Choloroacetate esterase (specific)
Nonspecific esterase

5. 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

7. M1 and M2

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

11. M3 M4 M5

12. Chloracetate (Specific) Esterase
Myeloid Cell Line
Naphthol-ASD-chloracetate
CAE > Free naphthol compounds
+ Stable diazonium salt (eg, Fast Corinth)
> Red deposit

13. Non-Specific Esterase
Monocytic Line
 Naphthyl acetate
ANAE > Free naphthyl compounds
+Stable diazonium salt (eg, Fast blue RR)
> Brown deposits

14. NSE with Fl inhibition
Histiocyte
Double Esterase in M4

15. FAB vs WHO Classifications of Hematologic Neoplasm
FAB criteria
Morphology
Cytochemistry
WHO criteria
Morphology
Immunophenotyping
Genetic features
Karyotyping
Molecular testing
Clinical features

16. WHO Classification of AML
AML with recurrent cytogenic translocations
AML with multi-lineage dysplasia
AML and myelodysplasia, therapy related
AML, not otherwise categorized

17. 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)

21. 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

22. L2 L3

23. Periodic Acid Schiff
Periodic acid + Glycogen
oxidation > Aldehyde + Schiff reagent
(para-rosaniline, Na metabisulfite)
> Red deposit

24. 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)

25. 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)

26. Antigens
B-Lineage
T-Lineage
HLA Dr
Tdt
CD34 +
0 to +
CD19
C 22
CD10
CD20
Cyt-
SIg
cALL and older
Pre-B and older
B-ALL
Pre-T
CD7
CD3
CD5
CD2
CD1
T-ALL

27. 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 during induction
Mkd reduction at day 7
Mild reduction at day 7

28. Typical Labs in CML Leukocytosis with blastemia Thrombocytosis
Basophilia
Micro-megakaryocytes
Low LAP score (intermediate if infected)
About 10% blasts in BM
Philadelphia chromosome

29. CML

30. Bone marrow aspirate
and biopsy

31. Pseudo-Gaucher’s cells in BM

32. Leukocyte Alkaline Phosphatase (LAP)
Naphthol AS-MX phosphate LAP at pH8.6 >
Naphthol AS-MX + Diazonium salt
(eg, Fast blue RR)
> Insoluble pigment

33. 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

34. 1+ 2+ 3+ 4+

35. LAP Score Example: 0 x 35 cells = 0 1+ x 30 cells = 30

36. 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

37. Karyotype 46,XX,t(9;22)(q34;q11.2) -- Ph chromosome

39. FISH showing the BCR (green), ABL (orange), and BCR-ABL fusion signals (arrow): A=positive (contains a residual ABL signal), B=normal

40. 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

41. 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

42. Myelofibrosis

43. Myelofibrosis

45. 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

46. CLL
PB and BM

49. Bone Marrow

51. WHO Lymphoid Neoplasms
B cell neoplasms
T/NK cell neoplasms
Hodgkin lymphoma (disease)

52. Mature B Cell Neoplasms
B cell CLL/SLL
B prolymphocytic leukemia
Burkitt’s lymphoma / leukemia
Splenic marginal zone B lymphoma
Extranodal marginal B lymphoma
Hairy cell leukemia
Lymphoplasmocytic leukemia
Mantle cell lymphoma
Plasma cell myeloma / plasmacytoma
Follicular lymphoma
Diffuse large B lymphoma

53. T/NK Cell Neoplasms T prolymphocytic leukemia
T granular lymphocytic leukemia
Aggressive NK cell leukemia
Adult T lymphoma / leukemia
Mycosis fungoides (Sezary syndrome)
Anaplastic large cell lymphoma
Hepatosplenic T lymphoma
Peripheral T lymphoma
Immunoblastic T lymphoma