The Acute Leukaemias
What Is Acute Leukemia?
• The acute leukaemias are a heterogeneous group of malignant disorders,
which are characterized b...
Hematopoietic
stem cell
Neutrophils
Eosinophils
Basophils
Monocytes
Platelets
Red cells
Myeloid
progenitor
Lymphoid
progen...
Myeloid maturation
myeloblast promyelocyte myelocyte metamyelocyte band neutrophil
MATURATIONMATURATION
Adapted and modifi...
Acute Leukemia
• accumulation of blasts in the marrow
Marrow failure
• neutropenia: infections, sepsis
• anemia: fatigue, pallor
• thrombocytopenia: bleeding
Demographics of Leukemia
Patients (2001 Data)
ALL
11%
CLL
26%
AML
31%
CML
15%
others
17%
Total Reported Cases = 31,500
Sou...
Acute Leukaemogenesis
• The acute leukaemias are thought to develop as a
result of a genetic alteration within a single st...
differentiation
block
enhanced
proliferation
Acute
Leukemia+
Gain of function mutations of
tyrosine kinases
eg. FLT3, c-KI...
Development of Leukemia in
the Bloodstream
Stage 1- Normal Stage 2- Symptoms Stage 3- Diagnosis
Stage 4- Worsening
Stage 5...
Lab evaluation
– The lab diagnosis is based on two things
– Finding a significant increase in the number of
immature cells...
Classification of the immature cells involved may be
done by:
1- Morphology – an experienced morphologist can
look at the ...
Myeloblasts with auer rods
Morphology, cont.
• ALL – in contrast to the myeloblast, the lymphoblast is a small
blast with scant cytoplasm, dense chro...
FAB
• In an attempt to improve the reproducibility and
comparability of the classification process, a grouped
of expert ha...
Classification of leukemia
Main classification
Chronic leukemia Acute leukemia
Lymphoid LymphoidMyeloid Myeloid
FAB
AML
M0...
FAB Characterization for ALL
• L1: Homogenous population of small lymphoblasts
with scanty cytoplasm and scanty nucleoli. ...
Acute Non-Lymphoblastic Leukaemia
Diagnosis Alternative Bone Marrow Appearance
M0 Identified by ultrastructural myeloperox...
Acute leukemias
• Acute lymphoblastic leukemia –
– They may be classified on the basis of the
cytological features of the ...
ALL-L1 (peripheral Blood)
ALL-L1 (Bone Marrow)
ALL-L2 (peripheral Blood)
ALL-L2 (Bone Marrow)
Acute leukemias
• L3 – This is the rarest form of ALL. The cell size
is large, with fine, homogenous nuclear
chromatin con...
ALL-L3 (peripheral Blood)
ALL-L3 (Bone Marrow)
Acute leukemias
– Incidence – ALL is primarily a disease of
young children (2-5 years), but it can also
occur in adults
– ...
Acute leukemias
– Prognosis – age, WBC count, and cell type are the
most important prognostic indicators
• Patients younge...
Acute leukemias
• Acute myeloid leukemia (also called acute
granulocytic leukemia) – classification
depends upon
– Bone ma...
AML – M1
• Note the myeloblasts and the auer rod:
Acute leukemias
• M2 – myeloblastic with maturation
– The bone marrow shows 30-89% blasts and > 10%
promyelocytes;
– This ...
AML – M2
• Note myeloblasts and hypogranulated
PMNs:
AML – M3
• Note hypergranular promyelocytes:
Acute leukemias
• M3m – hypogranular promyelocytic –
– The bone marrow has > 30% blasts
– The WBC count is increased.
– Li...
AML – M3m
• Note hypogranular promyelocytes:
AML – M4
• Note monoblasts and promonocytes:
Acute leukemias
• M5 – acute monoblastic leukemia
– >80% of the nonerythroid cells in the bone marrow are monocytic
– Ther...
AML – M5A
• Note monoblasts:
AML-M5B
• Note monoblasts, promonocytes, and
monocytes:
AML – M6
• Note M1 type monoblasts
Acute leukemias
• M7 - Acute megkaryoblastic leukemia
– This is a rare disorder characterized by extensive
proliferation o...
2- The Cytochemistry
______________________ALL____________AML__________
• Myeloperoxidase - +
• Suddan black - +
• Non-spe...
3- The Immunological
• Immunologic markers (immunophenotyping) – these are
used mainly for lymphocytes, i.e., for determin...
Direct versus indirect labeling
of antigens
Flow cytometer
Terminal deoyxtidyl transferase
• This is a unique DNA polymerase
present in stem cells and in precursor B
and T lymphoid ...
3- Classification upon The Immunological
Approach
• First: The AML FAB classification :
It has revealed that the blasts ar...
Second: The ALL-FAB classification is:
• Precursor B-ALL which is CD19+, cytoplasmic CD22+
and TdT+, includes three subtyp...
B cell maturation
T cell maturation
4- Cytogenetics
• Cytogenetics – cytogenetics studies can now be used for
diagnosis and for prognosis of hematologic
malig...
Chromosomal translocation
Chromosome karyotyping
Type of
Leukaemia
Chromosomal Abnormality FAB Type
ALL t (9;22) L1,L2
Pre-B ALL t (1;19) L1
B ALL t (8;14)
t (8;22)
t (2;8...
Pathophysiology of the Acute
Leukaemias
Acute leukaemia causes morbidity and mortality
through three general mechanisms:
•...
First: Deficiency in normal blood cell
number or function
• Infection
• Haemorrhage
• Anaemia
Constitutional symptoms
• fever and sweats common
• weight loss less common
2nd
:Infiltration of tissues/organs
• enlargement of liver, spleen, lymph
nodes
• gum hypertrophy
• bone pain
• other orga...
Gum hypertrophy
A
B
C
Chloromas
NEJM 1998
Leukostasis
• Leukostatic tumours accumulation of
blasts in microcirculation with impaired
perfusion
• lungs: hypoxemia, p...
Sanctuary site relapse
• Leukaemic infiltration of the testes and
meninges provide an effective
sanctuary for resident lae...
3rd
:Metabolic Disturbances
• hyponatraemia is relatively common in ANLL secondary to the
production of a vasopressin-like...
Laboratory features
• WBC usually elevated, but can be
normal or low
• blasts in peripheral blood
• normocytic anemia
• th...
Bone marrow in acute
leukemia
• necessary for diagnosis
• useful for determining type
• useful for prognosis
• Acute leuke...
Distinguishing AML from ALL
• light microscopy
– AML: Auer rods, cytoplasmic granules
– ALL: no Auer rods or granules.
• f...
AML
AML
Auer rods in AML
ALL
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The acute-leukaemias

  1. 1. The Acute Leukaemias
  2. 2. What Is Acute Leukemia? • 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. • 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.
  3. 3. Hematopoietic stem cell Neutrophils Eosinophils Basophils Monocytes Platelets Red cells Myeloid progenitor Lymphoid progenitor B-lymphocytesB-lymphocytes T-lymphocytes Plasma cells naïve ALLALL AMLAML
  4. 4. Myeloid maturation myeloblast promyelocyte myelocyte metamyelocyte band neutrophil MATURATIONMATURATION Adapted and modified from U Va website
  5. 5. Acute Leukemia • accumulation of blasts in the marrow
  6. 6. Marrow failure • neutropenia: infections, sepsis • anemia: fatigue, pallor • thrombocytopenia: bleeding
  7. 7. Demographics of Leukemia Patients (2001 Data) ALL 11% CLL 26% AML 31% CML 15% others 17% Total Reported Cases = 31,500 Sources from Leukemia, Lyphoma, Myeloma Facts 2001 CLL=Chronic Lymphocytic ALL=Acute Lymphocytic CML=Chronic Mylogenous AML=Acute Mylogenous
  8. 8. Acute Leukaemogenesis • 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. • 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.
  9. 9. differentiation block enhanced proliferation Acute Leukemia+ Gain of function mutations of tyrosine kinases eg. FLT3, c-KIT mutations N- and K-RAS mutations BCR-ABL TEL-PDGFβR Loss of function of transcription factors needed for differentiation eg. AML1-ETO CBFβ-SMMHC PML-RARα Two-hit model of leukemogenesis
  10. 10. Development of Leukemia in the Bloodstream Stage 1- Normal Stage 2- Symptoms Stage 3- Diagnosis Stage 4- Worsening Stage 5a- Anemia Stage 5b- Infection Legend White Cell Red Cell Platelet Blast Germ Sources from Leukemia, by D. Newton and D. Siegel
  11. 11. Lab evaluation – The lab diagnosis is based on two things – Finding a significant increase in the number of immature cells in the bone marrow including blasts, promyelocytes, promonocytes (>30% blasts is diagnostic) – Identification of the cell lineage of the leukemic cells – Peripheral blood: • Anemia (normochromic, normocytic) • Decreased platlets • Variable WBC count – The degree of peripheral blood involvement determines classification: » Leukemic – increased WBCs due to blasts » Subleukemic – blasts without increased WBCs » Aleukemic – decreased WBCs with no blasts
  12. 12. 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 and the presence of auer rods (are a pink staining, splinter shaped inclusion due to a rod shaped alignment of primary granules found only in myeloproliferative processes) to identify the blast type: • AML – the myeloblast is a large blast with a moderate amount of cytoplasm, fine lacey chromatin, and prominent nucleoli. 10-40% of myeloblasts contain auer rods.
  13. 13. Myeloblasts with auer rods
  14. 14. 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
  15. 15. 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.
  16. 16. Classification of leukemia Main classification Chronic leukemia Acute leukemia Lymphoid LymphoidMyeloid Myeloid FAB AML M0 M1 M2 M3 M4 M5 M6 M7 L1 L2 L3
  17. 17. 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.
  18. 18. 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 > 80% monoblasts is poorly differentiated (M5a) > 80% monoblasts, promonocytes or monocytes is well differentiated (m5b) 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.
  19. 19. 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. • L2 – This is the most frequent ALL found in adults. The cell size is large and heterogenous 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.
  20. 20. ALL-L1 (peripheral Blood)
  21. 21. ALL-L1 (Bone Marrow)
  22. 22. ALL-L2 (peripheral Blood)
  23. 23. ALL-L2 (Bone Marrow)
  24. 24. 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.
  25. 25. ALL-L3 (peripheral Blood)
  26. 26. ALL-L3 (Bone Marrow)
  27. 27. 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
  28. 28. 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%
  29. 29. 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: • M1 – myeloblastic without maturation – The bone marrow shows ≥ 90% blasts and < 10% promyelocytes – The disease occurs in older adults
  30. 30. AML – M1 • Note the myeloblasts and the auer rod:
  31. 31. 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 • 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
  32. 32. AML – M2 • Note myeloblasts and hypogranulated PMNs:
  33. 33. AML – M3 • Note hypergranular promyelocytes:
  34. 34. 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 and – It is characterized by a 15,17 translocation • 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
  35. 35. AML – M3m • Note hypogranular promyelocytes:
  36. 36. AML – M4 • Note monoblasts and promonocytes:
  37. 37. 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) • 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
  38. 38. AML – M5A • Note monoblasts:
  39. 39. AML-M5B • Note monoblasts, promonocytes, and monocytes:
  40. 40. AML – M6 • Note M1 type monoblasts
  41. 41. Acute leukemias • M7 - Acute megkaryoblastic leukemia – This is a rare disorder characterized by extensive proliferation of megakaryoblasts, atypical megakaryocytes and thrombocytopenia
  42. 42. 2- The Cytochemistry ______________________ALL____________AML__________ • Myeloperoxidase - + • Suddan black - + • Non-specific esterase - + in M4, M5 • Periodic acid Schiff (PAS) + in c-ALL + in M6 • Acid phosphatase + in T-ALL + in M6
  43. 43. 3- The Immunological • Immunologic markers (immunophenotyping) – these are used mainly for lymphocytes, i.e., for determining B cell or T cell lineage. These tests rely on antibodies made against specific surface markers. 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.
  44. 44. Direct versus indirect labeling of antigens
  45. 45. Flow cytometer
  46. 46. 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.
  47. 47. 3- Classification upon The Immunological Approach • First: The AML FAB classification : It has revealed that the blasts are usually CD13+, CD33+ and TdT-. Special antibodies are helpful in AML M6 and M7which show that M6 has glycophorin A + and M7 has CD41,42,61+.
  48. 48. 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.
  49. 49. B cell maturation
  50. 50. T cell maturation
  51. 51. 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.
  52. 52. Chromosomal translocation
  53. 53. Chromosome karyotyping
  54. 54. Type of Leukaemia Chromosomal Abnormality FAB Type ALL t (9;22) L1,L2 Pre-B ALL t (1;19) L1 B ALL t (8;14) t (8;22) t (2;8) L3 T ALL t (11;14) L1,L2 AML t (8;21) M2 APL t (15;17) M3 AmoL del/t (11) M5 AMMoL inv16,and t (16;16) M4 AEL t (8;16) M6 4- Cytogenetics
  55. 55. 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.
  56. 56. First: Deficiency in normal blood cell number or function • Infection • Haemorrhage • Anaemia
  57. 57. Constitutional symptoms • fever and sweats common • weight loss less common
  58. 58. 2nd :Infiltration of tissues/organs • enlargement of liver, spleen, lymph nodes • gum hypertrophy • bone pain • other organs: CNS, skin, testis, any organ
  59. 59. Gum hypertrophy
  60. 60. A B C Chloromas NEJM 1998
  61. 61. 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 )
  62. 62. 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.
  63. 63. 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.
  64. 64. Laboratory features • WBC usually elevated, but can be normal or low • blasts in peripheral blood • normocytic anemia • thrombocytopenia • neutropenia • DIC
  65. 65. 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)
  66. 66. Distinguishing AML from ALL • light microscopy – AML: Auer rods, cytoplasmic granules – ALL: no Auer rods or granules. • flow cytometry • special stains (cytochemistry)
  67. 67. AML
  68. 68. AML
  69. 69. Auer rods in AML
  70. 70. ALL
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