acute leukemias for undergraduates

1. LEUKEMIAS
Dr SAPNA M

2. Leukemoid Reaction
• A leukemoid reaction describes a high
WBC count with neutrophilia,usually in
response to infection.
• The WBC count may be as high as 50,000
/microL and can easily mimic CML or
AML.

3. • Serum leukocyte alkaline phosphatase is
normal or elevated in leukemoid reaction,
but is depressed in chronic myelogenous
leukemia.
• The bone marrow in a leukemoid reaction,
if examined, may be hypercellular but is
otherwise typically unremarkable.

4. Features Suggesting Leukemoid
Reaction
• Toxic granulation.
• High LAP score.
• Presence of an obvious cause for the
neutrophilia.

5. • As noted above, a leukemoid reaction is typically a response to an underlying medical issue. Causes of leukemoid reactions include:
• Hemorrhage
• Drugs
– Use of Sulfa drugs
– Use of Dapsone
– Use of glucocorticoids
– Use of G-CSF or related growth factors
– All-trans retinoic acid (ATRA)
• Infections
– Clostridium difficile
– Tuberculosis
– Pertussis
– Infectious mononucleosis (lymphocyte predominant)
– Visceral Larva Migrans (eosinophil predominant)
• Asplenia
• Diabetic ketoacidosis
• Organ necrosis
– Hepatic necrosis
– Ischemic colitis
• As a feature of Trisomy 21 in infancy (incidence of ~10%)
• As a paraneoplastic phenomenon (rare)

6. Leukemoid reaction

7. LAP score

9. What is leukemia?
Leukemia is cancer of the white blood cells. White
blood cells help your body fight infection. Your
blood cells form in your bone marrow. In leukemia,
however, the bone marrow produces abnormal
white blood cells. These cells crowd out the healthy
blood cells, making it hard for blood to do its work.

10. Hematopoietic
stem cell
Neutrophils
Eosinophils
Basophils
Monocytes
Platelets
Red cells
Myeloid
progenitor
Lymphoid
progenitor
B-lymphocytes
T-lymphocytes
Plasma
cells
naïve
ALL
AML

11. Myeloid maturation
myelobl
ast
promyelocytemyelocytemetamyelocyteband neutrophil
MATURATION
Adapted and modified from U Va website

12. Definition
• Leukemia: is a cancer of the blood or bone marrow
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.

13. About the Disease
• Leukemia, lymphoma and myeloma are
cancers that originate in the bone marrow
(leukemia & myeloma) or in lymphatic
tissues (lymphoma).

14. Different Types of Blood
Cancers
• Leukemia
• Non-Hodgkin Lymphoma
• Hodgkin Lymphoma
• Myeloma
• Myelodysplastic Syndromes

15. What is Leukemia
• Greek word which means “white blood”
• Leukemia is when cells spread rapidly and destroy living
tissue.
• It grows/invades the bone marrow which is the factory of
blood and replaces normal blood elements with cancer
cells.
• Cancer cells replace all bone marrow cells which causes
infection and bleeding problems.
• Leukemia is basically white blood cells that don‟t work
well and cause trouble.

16. Signs and Symptoms
• 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.

18. Causes
• Leukemia,like other malignancies, results
from somatic mutations in the DNA.
• Certain mutations produce leukemia by
activating oncogenes or deactivating
tumor suppressor genes.
• These mutations may occur spontaneously
or as a result of exposure to radiation
or carcinogenic substances,and likely
to be influenced by genetic factors.

19. Causes-cont‟d
• Ionizing radiation
• Viruses: Human T-lymphotropic virus (HTLV-1)
• Chemicals: Benzene,chemotherapy.
• Smoking: slight increase in leukemia
incidence.
• Genetic predisposition toward developing
leukemia: Down syn.,Fanconi anemia

21. Classification
• Multiple classification systems.
• FAB classification:
French-American-British Classification.
• FAB Classification relies on morphologic,
cytochemical,and immunophenotyping
criteria to define 8 major subtypes
(M0-M7)

22. FAB vs WHO Classification
• French-American-British (FAB) Cx
– Cellular morphology and cytochemical stain
– Acute leukemia as > 30% bone marrow blasts
– Widely used
• World Health Organization Cx
– Cellular morphology and cytochemical stain
– Immunologic probes of cell markers, cytogenetics,
molecular abnormalities & clinical syndrome
– Acute leukemia as > 20% bone marrow blasts
– Standard for diagnosis

23. Acute myeloid leukemias (AML)
Classification - FAB
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

24. AML classification - WHO
AML not otherwise categorized
1. AML minimally differentiated
2. AML without maturation
3. AML with maturation
4. Acute myelomonocytic leukemia
5. Acute monocytic leukemia
6. Acute erythroid leukemia
7. Acute megakaryocytic leukemia
8. Acute basophilic leukemia
9. Acute panmyelosis with myelofibrosis

27. Acute vs Chronic Leukemia
Acute Chronic
Age Children & young
adults
Middle age and
elderly
Onset Sudden insidious
Duration weeks to months years
WBC count Variable High

28. Acute vs Chronic Leukemia
Acute Chronic
Platelets Low Early: Normal/ High
Late: Low
Anemia High (>90%) None/ mild
Predomi-
nant cells
Blast cells Mature cells
AML = myeloblast
ALL= lymphoblast
CML=granulocytes
CLL=lymphocytes

29. Acute vs Chronic Leukemia
Acute Chronic
Marrow
cellularity
>20% marrow
blasts (WHO)
> 30% marrow
blasts (FAB)
>70% marrow
cellularity
(hypercellular);
No dysplasia
Diagnosis PBS, BM exam,
cytochemical
stains, surface
markers,
EM,chromosome
PBS (peripheral
blood smear)

30. Acute Myeloid Leukemia

31. Definition
• Acute myeloid leukemia (AML): acute
myelogenous leukemia,acute non-
lymphocytic leukemia.
• AML consists of a group of relatively well-
defined hematopoietic neoplasms
involving precursor cells commited
to the myeloid line(WBCs,RBCs,PLTs)

32. Chracteristics
• AML is characterized by a clonal proli-
feration of myeloid precursors with a
reduced capacity to differentiate into
mature cellular elements.
• As a result,there is an accumulation of
leukemic blasts or other immature
forms in the BM,peripheral blood,and
other tissues with a variable reduction in
the production of normal RBCs,platelets,
and mature granulocytes.

33. Specific:
• M2 : Chloroma:-presents as a mass lesion „tumor
of leukemic cells‟
• M3 : DIC
• M4/M5 : Infiltration of soft tissues,
gum infiltration, skin deposits ,Meningeal
involvement-headache, vomiting, eye symptoms

34. A
B
C
Chloromas
NEJM 1998

35. Gum hypertrophy

36. Leukostasis
• accumulation of blasts in microcirculation
with impaired perfusion
• lungs: hypoxemia, pulmonary infiltrates
• CNS: stroke
• only seen with WBC >> 50 x 109/L

37. Pathological Features
• CBC and differential.
• Blood film (smear).
• Bone marrow examination: BM aspirate
and trephine biopsy.
1-Morphology.
2-Immunephenotyping.
3-Cytogenetics and molecular biology.

38. Jemshidi trephine &
Salah aspiration needle

40. Bone marrow in acute leukemia
• necessary for diagnosis
• useful for determining type
• useful for prognosis
• Acute leukemias are defined by the
presence of > 20% blasts in bone marrow
(% of nucleated marrow cells)

41. • Bone marrow aspirate & trephine:
Hypercellular,
– blast cells ( > 20%),
– presence of Auer rods - AML type
• Cytochemistry :
Special stains to differentiate AML from
ALL ;
Positivity with Sudan black &
Myeloperoxidase (MPO) in AML

42. WBC Count in AML
• WBC count in AML can be high,normal,or
low.
• Median WBC count in AML is 15 000/uL.
• 20% of patients have > 100 000/uL
• 25-40% of patients have <5000/uL
• 95% of patients have blast cells on blood
film.

43. Distinguishing AML from ALL
• light microscopy
– AML: Auer rods, cytoplasmic granules
– ALL: no Auer rods or granules.
• flow cytometry
• special stains (cytochemistry)

44. Cytochemical Stains
• Since the early 20th century, cytochemical staining of
cells has been a useful tool in differentiating
hematopoietic diseases.
• Smears and imprints made from bone marrow, lymph
nodes, spleen, or peripheral blood are preferred.
– In enzymatic techniques, fresh smears are used to
ensure optimal enzyme activity
• Certain elements may be inhibited during the fixation
of smears and imprints

46. Myeloperoxidase (MPX/MPO)• The proxidase enzyme reacts with H2O2 & release O2,
which oxidizes the indicator dye and produce orange-
brown granules in the cells (3-amino-9-erythrocarbazol)
• Enzyme MPX is found in the 1o granules of
granulocytes, neutrophils and precursors (from the
promyelocyte stage on) & eosinophils
• Monocytes may be weakly pos
• Leukemic myeloblasts are usually pos and Auer rods
stain very strongly
• Used for differentiating AML (+) from ALL (-)
• Normal bone marrow smear <5 days old used for control
slides (promyelocyte - neutrophils)

47. MPO (right) & Sudan black (left)
showing intense localised positivity in
blasts

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

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

50. Non-Specific Esterase (NSE)
• Nonspecific esterase liberates alpha-naphthyl from
the substrate alpha-naphthyl acetate. Alpha-
naphthyl is couples with the dye molecule to form
dark reddish-brown granules
• Monocytes, monblasts, macrophages, histiocytes,
megakaryocytes and some carcinomas are NSE pos
• Abnormal erythroblasts are strongly pos
• Lymphocytes are neg or may show dot positivity

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

52. NSE continued
• Used for differentiating myelomonocytic and
monocytic leukemia (+) from granulocytic leukemia (-
)
• Monocyte NSE are fluoride sensitive
• Peripheral smear with appreciable # of monocytes or a
normal BM smear used for control slides
http://www.healthsystem.virginia.edu/internet/hematology/hessedd/malignanthematologicdisorders/leukemias/aml-m4.cfm

53. Double Esterase in M4
NSE with Fl inhibition
Histiocyte

54. Periodic Acid Schiff (PAS)
• Periodic acid oxidizes glycogen, mucoproteins, and
other high-molecular weight carbohydrates to
aldehydes.
• Aldehydes react with colorless Schiff reagent,
staining them a bright red-pink
• Megakaryocytes and platelets stain strongly pos
• Normoblasts will stain Pos
• Lymphoblasts in ALL show course and granular
(block) positivity

55. PAS Continued
• Myeloblasts are Neg
• Aids in diagnosis of ALL, erythroleukemia, and
megakaryoblastic leukemia
• Normal bone marrow smear used for control slides
http://www.pathologyoutlines.com/leukemia.html

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

57. AML

58. AML

59. Auer rods in AML

60. ALL

61. P. Smear AML

62. MO: Minimally differentiated
• Undifferentiated Blasts
(No maturation)
• Myeloid phenotype -
CD13, CD33, CD34
• (-) SBB, MPO
• Negative: Auer rods,
Esterase

63. M1 AML without maturation
 > 30% myeloblasts
 Large cells, round nucleus
 Nucleoli (+)
 scanty cytoplasm
 >3% MPO, SBB (+)
 <20% NSE (+)
 CD 13, 33, 117

64. M1 AML without maturation

65. M2 AML with maturation
• Common type
• >30% myeloblasts
• >10% granulocyte
• Kidney shape nucleus
• Nucleoli (+)
• (+) Auer rods
• Eosinophilic granules
• >50% MPO, SBB (+)
• CD 13, 33

66. M2 AML with maturation

67. Auer Rods

68. M3 (hypergranular promyelocytic)
• Promyelocyte-predominant
• Large, kidney shape
• (+) Auer rods (faggot cells)
• basophilic, bilobed nuclei
• CD 13,33
• High incidence of DIC

69. Acute myeloid leukemia with very
abnormal cells (AML M3/ t15;17)

70. M4 Acute myelomonocytic
• >30% myeloblast (FAB)
• >20% granulocyte
• >20% promonocytes and
monocytes
• CD 11, 13, 33,14
• (+) Auer rods common
• High serum lysozyme level
– M4Eo = w/ eosinophilia

71. M3
M5
M4

72. M5: acute monocytic leukemia
1. M5a – without maturation
– Monoblasts , few promonocytes
2. M5b – with maturation
– Blast, Promonocytes (BM), Monocytes
(Blood)

74. M5a
• Monoblast ameboid with
round to oval nuclei,
• prominent nucleoli,
• <20% promonocytes/mono
• Vacuolated cytoplasm

75. AML M5a

76. M5b
• > 20% promonocytes,
monocytes
• Promonocytes folded,
convulated nucleus
• Azurophilic granules

77. AML M5b

78. M6 - erythroleukemia
 Large, bizarre,
round-to-oval cells
 (+) nucleoli
 > 50% Erythroblasts
 > 30 % Myeloblasts
 CD 45,71 Glycophorin A
 CD 13, 15,33 myeloblast
 PAS (+)

79. M6 (erythroblast)

80. M6 (erythroblast)

81. M7 – acute megakaryoblastic
• >30% megakaryoblasts
• platelet like granules on
PAS stain
• NSE (but not BE) (+)
• Myeloid blasts may show
SBB or MPO (+)
• CD 41,42,61

82. M7 (Megakaryoblast)

83. Megakaryoblast

84. Acute Nonlymphocytic Leukemias

85. • Acute lymphoblastic leukemias

86. 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

87. 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)

88. • Confirmation:
– Immunophenotyping
– Molecular genetics
– Cytogenetics: Chromosomal abnormalities

89. ALL L1

90. L3L2

91. Burkits / ALL L3

92. Prognostic factors
• High WBC – relapse in testis /cns
• Infants <1 yr, children >10 yr poor outcome
• L1- good prognosis
• L2,3- bad prognosis

93. Prognosis
• The response to treatment and overall
survival of patients with AML are
heterogenous.
• Prognostic factors are related to patient
and tumor characteristics:
1-Age
2-Performance status
3- Karyotype

94. Adverse Clinical Predictors
• Advanced age.
• Poor performance status.
• History of exposure to cytostatic agents or
radiotherapy.(Therapy-related AML).
• History of MDS or other hematological
diseases

95. THANK YOU