1. Ahmad Sh. Silmi Msc Haematology, FIBMS
The Acute Leukaemias
Ahmad Sh. Silmi
Msc Haematology, FIBMS

2. What Is Acute Leukemia?(1)
The acute leukaemias are a heterogeneous group of malignant disorders, which are characterized by the uncontrolled clonal proliferation and accumulation of poorly differentiated blast cells in the bone marrow and other tissues.
Thus replacement of normal bone marrow elements with abnormal (neoplastic) blood cells.

3. What Is Acute Leukemia?(2)
These leukaemic cells are frequently (but not always) present in the peripheral blood stream. Subsequently there is a raised total blood count and evidence of bone marrow failure (i.e. anaemia, neutoropenia, thrombocytopenia) are ensues.
In the acute leukaemias the blasts commonly invade reticuloendothelial tissues including the spleen, liver and lymph nodes. They may also invade other tissues, infiltrating any organ of the body.
If left untreated, leukaemia eventually causes death.

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

5. Myeloid maturation MATURATION myeloblast promyelocyte myelocyte
metamyelocyte
band
neutrophil
MATURATION
Adapted and modified from U Va website 5

6. Acute Leukemia
accumulation of blasts in the marrow 6

7. Marrow failure Neutropenia: infections, sepsis Anemia: fatigue, pallor
Thrombocytopenia: bleeding 7

8. Demographics of Leukemia Patients (2001 Data)
CLL=Chronic Lymphocytic
ALL=Acute Lymphocytic
CML=Chronic Mylogenous
AML=Acute Mylogenous
Total Reported Cases = 31,500
Sources from Leukemia, Lyphoma, Myeloma Facts 2001

9. Acute Leukaemogenesis (1)
The acute leukaemias are thought to develop as a result of a genetic alteration within a single stem cell in the bone marrow. Which means, a blockage of maturation at certain level; this reveals an abnormal regulatory mechanism, and the cell is incapable for further proliferation and differentiation, but it still has the ability to divide.

10. Acute Leukaemogenesis (2)
Successive mitotic division in the progeny of the cell produces a clonal population, which once it is sufficient a clonal mass has been achieved, and gives rise to the clinical manifestation of the disease.

11. Two-hit model of leukemogenesis
Loss of function of transcription factors needed for differentiation
eg. AML1-ETO
CBFb-SMMHC
PML-RARa
Gain of function mutations of tyrosine kinases
eg. FLT3, c-KIT mutations
N- and K-RAS mutations
BCR-ABL
TEL-PDGFbR
differentiation
block
enhanced
proliferation
Acute
Leukemia +

12. Development of Leukemia in the Blood stream
Stage 1- Normal
Stage 2- Symptoms
Stage 3- Diagnosis
Stage 5a- Anemia
Stage 5b- Infection
Stage 4- Worsening
Legend
White Cell
Red Cell
Platelet
Blast
Germ
Sources from Leukemia, by D. Newton and D. Siegel

13. Acute leukemias - laboratory findings
1. Blood examination
- anaemia.
- thrombocytopenia.
- variable leukocyte count, usually increased.
- blood morphology: presence of blast cells.
2. Bone marrow morphology
- presence of blast cells.
- suppression of normal hematopoiesis.

14. Acute leukemias - Laboratory findings (2)
3. Cytochemical stains
4. Immunophenotyping
5. Cytogenetics
6. Molecular studies

15. Classification of the immature cells involved may be done by:
1- Morphology – an experienced morphologist can look at:
the size of the blast.
the amount of cytoplasm.
the nuclear chromatin pattern.
the presence of nucleoli.
the presence of auer rods.
N.B: Auer rods are a pink staining, splinter shaped inclusion due to a rod shaped alignment of primary granules found only in myeloproliferative processes.

16. Myeloblasts with auer rods
AML – The myeloblast is a large blast with a moderate amount of cytoplasm, fine lacey chromatin, and prominent nucleoli % of myeloblasts contain auer rods.

17. Morphology, cont.
ALL – in contrast to the myeloblast, the lymphoblast is a small blast with scant cytoplasm, dense chromatin, indistinct nucleoli, and no auer rods
Lymphoblast

18. FAB
In an attempt to improve the reproducibility and comparability of the classification process, a grouped of expert haematologists from French, America, and Britian (FAB) collaborated to define a more objective criteria for the classification of acute leukaemias.
The initial FAB study was based on the examination of more than 200 different cases of acute leukaemia by expert morphologists, in addition, a myeloperoxidase or suddan black stains should be used to facilitate the recognition of myeloblasts.

19. Classification of leukemia
Main classification
Chronic leukemia
Acute leukemia
FAB
Lymphoid
Myeloid
Lymphoid
Myeloid L1 L2 L3
AML M0 M1 M2 M3 M4 M5 M6 M7

20. FAB Characterization for ALL
L1: Homogenous population of small lymphoblasts with scanty cytoplasm and scanty nucleoli. Nucleus occasionally cleft.
L2: Heterogeneous population of large lymphoblasts with moderately abundant cytoplasm & or more nucleoli. Nucleus commonly intended or cleft.
L3-Burkitt’s type: Homogenous population of large lymphoblasts with prominent nucleoli & deeply basophilic, vacuolated cytoplasm.

21. Morphologic subtypes of acute lymphoblastic leukemias (FAB classification)
Subtype Morphology Occurrence (%)
L Small round blasts
clumped chromatin
L2 Pleomorphic larger blasts
clefted nuclei, fine chromatin
L3 Large blasts, nucleoli,
vacuolated cytoplasm

22. Acute leukemias Acute lymphoblastic leukemia –
They may be classified on the basis of the cytological features of the lymphoblasts into:
L1 - This is the most common form found in children and it has the best prognosis. The cell size is small with fine or clumped homogenous nuclear chromatin and absent or indistinct nucleoli. The nuclear shape is regular, occasionally clefting or indented. The cytoplasm is scant, with slight to moderate basophilia and variable vacuoles.

23. ALL-L1 (peripheral Blood)

24. ALL-L1 (Bone Marrow)

25. ALL – L2
L2 – This is the most frequent ALL found in adults. The cell size is large and heterogeneous with variable nuclear chromatin and prominent nucleoli. The nucleus is irregular, clefting and indented. The cytoplasm is variable and often moderate to abundant, the basophilia is variable and may be deep, and vacuoles are variable.

26. ALL-L2 (peripheral Blood)

27. ALL-L2 (Bone Marrow)

28. Acute leukemias
L3 – This is the rarest form of ALL. The cell size is large, with fine, homogenous nuclear chromatin containing prominent nucleoli. The The nucleus is regular oval to round. The cytoplasm is moderately abundant and is deeply basophilic and vacuolated.

29. ALL-L3 (peripheral Blood)

30. ALL-L3 (Bone Marrow)

31. Acute leukemias
Incidence – ALL is primarily a disease of young children (2-5 years), but it can also occur in adults.
Clinical findings – pancytopenia with resulting fatigue, pallor, fever, weight loss, irritability, anorexia, infection, bleeding, and bone pain.
L1 occurs in children, L2 in adults, and L3 is called Burkitts leukemia.

32. Acute leukemias
Prognosis – age, WBC count, and cell type are the most important prognostic indicators
Patients younger then 1 and greater than 13 have a poor prognosis
If the WBC count is < 10 x 109/L at presentation, the prognosis is good; If the WBC count is > 20 x 109/L at presentation the prognosis is poor
T cell ALL (more common in males) has a poorer prognosis than any of the B cell ALLs which have a cure rate of 70%.

33. AML

34. Acute leukemias
Acute myeloid leukemia (also called acute granulocytic leukemia) – classification depends upon
Bone marrow blast morphology
Degree of cell maturation
Cytochemical stains
Immunophenotyping
AML is divided into 7 different classifications:

35. Acute Non-Lymphoblastic Leukaemia
Diagnosis
Alternative
Bone Marrow Appearance M0
Identified by ultrastructural myeloperoxidase activity or immunophenotyping. M1
AML without maturation
Monomorphic with one or more distinct nucleoli, occasional auer rod and at least 3% myeloperoxidase positivity. M2
AML with maturation
50% OR > myeloblasts & promyelocytes and common single auer rod. Dysplastic myeloid differentiation may also be present. M3
APL
Dominant cell type is promyelocyte with heavy azurophilic granulation. Bundles of Auer rods confirm diagnosis. Microgranular variant exist (M3v) M4
AMMoL
As M2 but > 20% promonocytes & monocytes. M5
AMoL
M5a:> 80% monoblasts is poorly differentiated.
M5b:> 80% monoblasts, promonocytes or monocytes is well differentiated. M6
AEL
>50% bizzar, dysplastic nucleated red cells with multinucleate forms and cytoplasmic bridging. Myeloblasts usually > 30%. M7
AMegL
Fibrosis, heterogeneous blasts population with cytoplasmic blebs. Platelet peroxidase positive.

36. AML - M1 M1 – myeloblastic without maturation
The bone marrow shows  90% blasts and < 10% promyelocytes
The disease occurs in older adults

37. AML – M1
Note the myeloblasts and the auer rod:

38. Acute leukemias M2 – myeloblastic with maturation
The bone marrow shows 30-89% blasts and > 10% promyelocytes;
This is characterized by an 8,21 chromosomal translocation
This occurs in older adults

39. AML – M2
Note myeloblasts and hypogranulated PMNs:

40. AML - M3 M3 – hypergranular promyelocytic
This form of AML has a bone marrow with >30% blasts
Is more virulent than other forms
Occurs with a medium age of 39
The WBC count is decreased
Treatment causes a release of the granules and may send the patient into disseminated intravascular coagulation and subsequent bleeding
It is characterized by a 15,17 chromosomal translocation

41. AML – M3
Note hypergranular promyelocytes:

42. Acute leukemias M3m – hypogranular promyelocytic –
The bone marrow has > 30% blasts
The WBC count is increased.
Like the M3 type, treatment causes a release of the granules and may send the patient into disseminated intravascular coagulation and subsequent bleeding.
It is characterized by a 15,17 translocation

43. AML – M3m
Note hypogranular promyelocytes:

44. AML-M4 M4 – acute myelomonoblastic leukemia
Both myeloblasts and monoblasts are seen in the bone marrow and peripheral blood
Infiltration of extramedullary sites is more common than with the pure granulocytic variants

45. AML – M4
Note monoblasts and promonocytes:

46. Acute leukemias M5 – acute monoblastic leukemia
>80% of the nonerythroid cells in the bone marrow are monocytic
There is extensive infiltration of the gums, CNS, lymph nodes and extramedullary sites
This form is further divided into
M5A - Poorly differentiated (>80% monoblasts)
M5B - Well differentiated (<80% monoblasts)

47. AML – M5A
Note monoblasts:

48. AML-M5B
Note monoblasts, promonocytes, and monocytes:

49. AML-M6 M6 – erythroleukemia
This is rare and is characterized by a bone marrow having a predominance of erythroblasts
It has 3 sequentially morphologically defined phases;
Preponderance of abnormal erythroblasts
Erythroleukemia – there is an increase in both erythroblasts and myeloblasts
Myeloblastic leukemia – M1, M2, or M4
Anemia is common

50. AML – M6
Note M1 type monoblasts

51. Acute leukemias M7 - Acute megkaryoblastic leukemia
This is a rare disorder characterized by extensive proliferation of megakaryoblasts, atypical megakaryocytes and thrombocytopenia.

52. 2- The Cytochemistry Cytochemistry AML ALL Myeloperoxidase + -
Sudan black
Non-specific esterase + (M4,5) -
PAS (M6) + c-ALL
Acid phosphatase + (M6) +T-ALL

53. Acute lymphoblastic leukemias - reactivity with special stains
Subtype Peroxidase or Non-specific Periodic
Sudan black esterase acid-Schiff L L L

54. 3- The immunophenotyping
Significance: used for determining B cell or T cell lineage.
Principle: antibodies made against specific surface markers.
Procedure: They constitute what we would call the primary antibody and in an indirect assay they are allowed to react with the cells and unbound antibody is then washed away. Fluorescently labeled antibody (secondary antibody) against the primary antibody is added and allowed to react and then unbound secondary antibody is washed away. The cells are then sent through a flow cytometer that will determine the number of cells that have a fluorescent tag and which are thus positive for the presence of the surface marker to which the primary antibody was made. In a direct assay, the primary antibody is fluorescently labeled.

55. Direct versus indirect labeling of antigens

56. Flow cytometer

57. Terminal deoyxtidyl transferase
This is a unique DNA polymerase present in stem cells and in precursor B and T lymphoid cells. High levels are found in 90% of lymphoblastic leukemias. It can also be detected using appropriate antibodies and flow-cytometry.

58. 3- Classification upon The Immunological Approach
First: The AML FAB classification :
CD13+, CD33+ and TdT-.
M6 has glycophorin A +
M7 has CD41,42,61+.

59. Second: The ALL-FAB classification is:
Precursor B-ALL which is CD19+, cytoplasmic CD22+ and TdT+, includes three subtypes, common-ALL which is CD10+, null type which is CD10- and pre-B-ALL which shows intracytoplasmic m chains ( and may be CD10+ 0r CD10- ).
B-ALL which shows surface immunoglobulin (Ig) and TdT-.
T-ALL which shows T-cell antigens CD7+, cytoplasmic CD3+ and TdT+. 
B-ALL usually corresponds to the morphological L3 type whereas the CD10+, null, pre-B or T types may all be L1 or L2 and are morphologically indistinguishable.

60. Immunologic classification of acute lymphoblastic leukemias
B- lineage (80%) Markers
Pro-B CD19(+),Tdt(+),CD10(-),CyIg(-).
Common CD19(+),Tdt(+),CD10(+),CyIg(-).
Pre-B CD19(+),Tdt(+),CD10(+),CyIg(+),SmIg(-). Mature-B CD19(+),Tdt(+),CD10(±),CyIg(±),SmIg(+).
T-lineage (20%)
Pre-T CD7(+), CD2(-), Tdt(+).
Mature-T CD7(+), CD2(+), Tdt(+).

61. B cell maturation

62. T cell maturation

63. 4- Cytogenetics
Cytogenetics – cytogenetics studies can now be used for diagnosis and for prognosis of hematologic malignancies.
Many leukemias (and lymphomas) are characterized by specific chromosomal abnormalities, including specific translocations and aneuploidy. The specific type of malignancy can be identified based on the specific abnormality or translocation. These may be identified by
Looking at the karyotypes of the chromsomes from the abnormal cells
DNA based tests – these tests are very useful for following the course of the disease
A normal karyotype is usually associated with a better prognosis.

64. Chromosomal translocation

65. Chromosome karyotyping

66. 4- Cytogenetics FAB Type Chromosomal Abnormality Type of Leukaemia
L1,L2
t (9;22 )
ALL L1
t (1;19 )
Pre-B ALL L3
t (8;14 )
t (8;22)
t (2;8 )
B ALL
t (11;14)
T ALL M2
t (8;21)
AML M3
t (15;17)
APL M5
del/t (11)
AmoL M4
inv16,and t (16;16)
AMMoL M6
t (8;16)
AEL

67. t(9;11) and other translocations involving 11q23 (MLL)
4- Cytogenetics M2
t(8;21) (q22;q22)
AML1(CBFa )/ETO M3
t(15;17)(q22;q12)
PML/RARa
M4Eo
Inv(16)(p13q22)
CBFb /MYH1 M5
t(9;11) and other translocations involving 11q23 (MLL)

68. Chromosomal/molecular abnormalities with prognostic significance in ALL
Better prognosis
- normal koryotype
- hyperdiploidy
Poor prognosis
- t (8; 14)
- t (4; 11)
Very poor prognosis
- t (9; 22); BCR/ABL (+)

69. Chromosomal/molecular abnormalities with prognostic significance in AML
Favorable
t(8;21), t(15;17), inv(16)
Intermediate (Most patients)
normal, +8, +21, +22, del(7q), del(9q),
Adverse
-5, -7, del(5q), abnormal 3q,
complex karyotype (> 3 -5 abnormalities)

70. Pathophysiology of the Acute Leukaemias
Acute leukaemia causes morbidity and mortality through three general mechanisms:
Deficiency in normal blood cell number or function.
Invasion of vital organs with impairment of organ function.
Systemic disturbances shown by metabolic imbalance.

71. First: Deficiency in normal blood cell number or function
Infection
Haemorrhage
Anaemia

72. Constitutional symptoms
fever and sweats common
weight loss less common 72

73. 2nd:Infiltration of tissues/organs
enlargement of liver, spleen, lymph nodes
gum hypertrophy
bone pain
other organs: CNS, skin, testis, any organ 73

74. Gum hypertrophy 74

75. Gingival Infiltration in Monocytic (AML M4 eos) Variant of AML
Mani, A, Lee, DA. Leukemic Gingival Infiltration. N Engl J Med 2008; 358(3): 274. Copyright ©2008 Massachusetts Medical Society

76. Chloromas A B C
NEJM 1998 76

77. Leukostasis
Leukostatic tumours accumulation of blasts in microcirculation with impaired perfusion
lungs: hypoxemia, pulmonary infiltrates
CNS: stroke
only seen with WBC >> 50 x 109/L (Hyperleucocytosis ) 77

78. Sanctuary site relapse
Leukaemic infiltration of the testes and meninges provide an effective sanctuary for resident laeukaemic blasts because cytotoxic drugs poorly penetrate them.
This provides a source for relapse.
Meningeal and testicular relapse are most commonly seen in childhood ALL.

79. 3rd:Metabolic Disturbances
hyponatraemia is relatively common in ANLL secondary to the production of a vasopressin-like substance by myeloblasts.
Hypokalaemia is also common, especially in AMMoL and AmoL secondary to the renal damage.
Spontaneous lysis of blast cells causes the release of abnormally large amounts of purines into the plasma. This results to an increase uric acid or hyperuricaemia. This abnormality may be severe enough to require dialysis, particularly where renal damage is present.
Several of the cytotoxic drugs are nephrotoxic and therefore may increase the severity of minor metabolic disturbances secondary to massive cell lysis.

80. Notes to Remember

81. Laboratory features WBC usually elevated, but can be normal or low
blasts in peripheral blood
normocytic anemia
thrombocytopenia
neutropenia
DIC 81

82. 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) 82

83. Distinguishing AML from ALL
light microscopy
AML: Auer rods, cytoplasmic granules
ALL: no Auer rods or granules.
flow cytometry
special stains (cytochemistry) 83

84. AML 84

85. AML 85

86. Auer rods in AML 86

87. ALL 87