Transcript Introduction to Leukaemia

Acute Leukaemia
Ahmad Shihada Silmi
Msc, FIBMS
Staff Specialist in Hematology
Medical Technology Department
Islamic University of Gaza
DEFINITION
Leukemia is a malignant disease
characterized by unregulated proliferation of
one cell type.
– It may involve any of the cell lines or a stem cell
common to several cell lines.
ALL
naïve
B-lymphocytes
Lymphoid
progenitor
Plasma
cells
T-lymphocytes
AML
Hematopoietic
stem cell
Myeloid
progenitor
Neutrophils
Eosinophils
Basophils
Monocytes
Platelets
Red cells
ACUTE vs CHRONIC LEUKAEMIA
• Poorly differentiated blast population
• Rapidly fatal outcome, if untreated
versus
• Well differentiated cell population
• Associated with longer survival, even if left
untreated
AML
vs.
CML
ACUTE LEUKAEMIA
–Is a result of:
Malignant transformation of a stem cell leading
to unregulated proliferation and
Arrest in maturation at the primitive blast
stage. Remember that a blast is the most
immature cell that can be recognized as
committed to a particular cell line.
Two-hit model of leukemogenesis
Loss of function of
transcription factors
needed for differentiation
eg. FLT3, c-KIT mutations
N- and K-RAS mutations
BCR-ABL
TEL-PDGFbR
eg. AML1-ETO
CBFb-SMMHC
PML-RARa
differentiation
block
Gain of function mutations of
tyrosine kinases
+
enhanced
proliferation
Acute
Leukemia
Classification of acute leukemias
ALL
mainly children
M>F
curable in 70% of
children
curable in minority of
adults
AML
mainly adults
M>F
curable in minority of
adults
AML Fast Facts

In 2008…(UK)
13,290 new cases
 8820 deaths


In comparison…
Lung cancer-220,000 new cases and 160,000 deaths
 Pancreas 42,000 new cases and 35,000 deaths
 Lymphoma-65,000 new cases and 19,000 deaths


Relatively rare, but devastating disease for which new,
less toxic therapies are needed
Acute myeloid leukemia age-specific incidence rates: 1998-2002
(NCI-SEER Program)
Tallman, M. S. Hematology 2005;2005:143-150
Copyright ©2005 American Society of Hematology. Copyright restrictions may apply.
Biology of Leukemia
Resistance to apoptosis (even when DNA is
damaged)
De-regulation of growth
Differentiation block
Clinical presentation:
Will present with sign or symptoms related to :
– Pancytopenia:
WBCinfection.
Hb anemia.
platelets bleeding.
– Organ infiltration:
Lymphadenopathy.
More common with ALL than AML.
Splenomegally.
Hepatomegally.
CNS:5-10% of patient with ALL
Acute Lymphoblastic Leukaemia
Cancer of the blood affecting the white blood
cell known as LYMPHOCYTES.
Commonest in the age 2-10 years
Peak at 3-4 years.
Incidence decreases with age, and a
secondary rise after 40 years.
In children - most common malignant
disease
85% of childhood leukaemia
Acute Lymphoblastic Leukemia
Specific manifestation :
*bone pain, arthritis
*lymphadenopathy
*hepatosplenomegaly
*mediastinal mass
*testicular swelling
*meningeal syndrome
Acute Myeloid Leukemia
Arise from the malignant transformation of
a myeloid precursor
Rare in childhood (10%-15%)
The incidence increases with age
80% in adults
Most frequent leukemia in neonate
Acute Myeloid Leukemia
Specific manifestation :
- Gum hypertrophy
– Hepatosplenomegaly
– Skins deposit
– Lymphadenopathy
– Renal damage
– DIVC
On presentation:
1012 leukemia cells (1 liter volume).
~70% of adults treated with induction
chemotherapy will achieve remission.
– Remission: Normal counts (platelets>100K,
ANC>1000, transfusion independent, blasts < 5%)
AML: Working hypotheses
AML is a stem cell disease:
– The “bulk” cells circulating in peripheral blood are mostly
non-dividing, partially differentiated
– Leukemia derived from more primitive (more multi-potent)
stem cells will be harder to cure.
– The features of the bulk cells offer clues as to how primitive
the leukemia stem cell is…
How much chemotherapy can you give?
– Not so much that the normal stem/progenitors in the body
(gut, marrow, etc…) can’t recover.
Lifespan
Hematopoietic
Stem Cell
Decades
Years
Months
Weeks, Days
Platelets
Red blood cells
White blood cells
Lifespan
Hematopoietic
Stem Cell
Decades
Years
Months
Weeks, Days
Platelets
Red blood cells
White blood cells
Lifespan
Hematopoietic
Stem Cell
Decades
Years
Months
Weeks, Days
Platelets
Red blood cells
White blood cells
Lifespan
Hematopoietic
Stem Cell
Decades
Years
Months
Weeks, Days
Platelets
Red blood cells
White blood cells
Lifespan
Hematopoietic
Stem Cell
Decades
Years
Months
Weeks, Days
Platelets
Red blood cells
White blood cells
Lifespan
Hematopoietic
Stem Cell
Decades
Years
Months
Weeks, Days
Platelets
Red blood cells
White blood cells
Hematopoietic
Stem Cell
Platelets
Red blood cells
White blood cells
Hematopoietic
Stem Cell
Platelets
Red blood cells
White blood cells
Hematopoietic
Stem Cell
Platelets
Red blood cells
White blood cells
Hematopoietic
Stem Cell
Acute
Leukemia
Hematopoietic
Stem Cell
Chemotherapy
Hematopoietic
Stem Cell
Residual leukemia
stem cells
Remission
Platelets
Red blood cells
White blood cells
AETIOLOGY
Ionizing Radiation
• Atomic Bombs
- Hiroshima and Nagasaki, Utah, Chernobyl,
Three Mile Island fall-out
- within 1.5 - 3 years
- peak incidence 5-10 years
- dose dependent
AETIOLOGY
• Occupational exposure
- radiologists
- nuclear industry
• Diagnostic radiographic procedures
- Thorium dioxide
- foetal exposure from radiographic
pelvimetry
• Natural background radiation (EM)
AETIOLOGY
• Chemicals
- benzene
- rubber industry
- pesticides
• Viruses
- EBV
- Human retroviruses (HTLV 1&2)
AETIOLOGY
Host factors •
- familial occurrence
- genetic disorders (Down’s syndrome)
AETIOLOGY
Medications •
- Chemotherapy (Alkylating agents)
within 4-5 years
proportionate to dose and duration
- Radiotherapy: adds extra risk
- Immunosuppression
CLASSIFICATION
OF ACUTE LEUKAEMIA
Based on cell origin
- myeloid: some features of granulocyte and/or
monocyte development
- lymphoid: from lymphocyte precursors
- ambiguous lineage: undifferentiated, bilineal
or biphenotypic
CLASSIFICATION OF ACUTE LEUKAEMIA
• Standardisation of treatment
• Some acute leukaemias have peculiar
features
- ALL and lymph node, thymus and CNS
involvement
- APML and bleeding
CLASSIFICATION OF ACUTE
LEUKAEMIA
• Peripheral blood morphology
• Bone marrow morphology
• Immunophenotyping
• Cytogenetics
• Molecular studies
MORPHOLOGY
Romanowsky stained smears of
peripheral blood or bone
marrow
Nuclear chromatin pattern is most
important
AML
delicate chromatin
much cytoplasm
fine granules/Auer rods
ALL
clumped chromatin
scant cytoplasm
no granules
BASIS OF FAB CLASSIFICATION
30 % blast in bone marrow
Morphology of blast – Romanowsky stain
Cytochemical Stains – Myeloperoxidase
– Non specific esterase
CLASSIFICATION OF ACUTE
LEUKAEMIAS
ALL
• ALL/L1: 80%
- small blast cells
- high N/C ratio
ALL/L2 : 20% •
- larger cells, heterogeneous
- lower N/C ratio
ALL/L1
ALL/L2
CLASSIFICATION OF ACUTE
LEUKAEMIAS
ALL
• ALL/L3 : 3-5%
- large, homogeneous cells
- fine chromatin, prominent nucleoli
- intensely basophilic cytoplasm,
vacuoles
ALL/L3
Prognosis in ALL
parameters
Good
poor
WBC
low
High(>50x10 9 /l)
Gender
Girls
Boys
Immunophenotype
C-ALL
B-ALL
Age
Child
Adult or infant.
Cytogenetic
Normal,hyperdiploid,
Ph+,11q23rearrangements.
Time to clear blast
from blood
< 1week
>1week
Time to remission
<4weeks
>4weeks
Cns disease at
presentation
Absent
Present
Minimal residual
disease.
Negative at 1-3 months
Still positive at 3-6 months.
CLASSIFICATION OF ACUTE
LEUKAEMIAS
AML
FAB
Morphology
MPO
SE NSE
Comment
M0
Undifferentiated
neg
neg neg
primitive (bad?)
M1
Min. different.
pos
neg neg
primitive (bad?)
M2
Differentiated
pos
pos neg
t8;21
M3
Differentiated
pos
pos neg
t15;17 (APL)
M4
Myelomonocytic
pos
pos pos
INV16/M4eo
M5
Monocytic
pos
neg pos
11q23, FLT3
M6
Erythroleukemia
neg
neg neg
BAD…
M7
Megakaryocytic
neg
neg neg
BAD…
CLASSIFICATION OF ACUTE
LEUKAEMIAS
AML
Granulocytic differentiation 60%
- M0 - Acute myeloblastic leukaemia
with minimal differentiation
- M1 - Acute myeloblastic leukaemia
with minimal maturation
- M2 - Acute myeloblastic leukaemia
with significant maturation
- M3 - Acute promyelocytic leukaemia
AML/M0
AML/M1
AML/M2
AML/M3
Monocytic differentiation
- M4 - Acute myelomonocytic leukaemia
25%
- M5 - Acute monocytic leukaemia 10%
Erythroid differentiation
- M6 - Acute erythroleukaemia <5%
Megakaryocytic differentiation
- M7 -Acute megakaryoblastic leukaemia
<5%
AML/M4
AML/M5
AML/M6
AML/M7
IMMUNOPHENOTYPING
objective
• Measured by flow cytometry as intensity
with which a fluorescence - conjugated
antibody stains cell surface proteins.
• Indicates lineage commitment
• Indicates position along the normal
haemopoietic pathway at the time of
transformation
procedure
Flow Cytometry:
– Sample of whole blood in green or purple top
– RBCs lysed, WBC pellet washed.
– Antibodies with fluorochrome labels added.
– Stained cells analyzed by FACS
– Whole process can be done in two hours.
Immunophenotyping
Immunophenotyping
MYELOID
CD13
LYMPHOID
B lineage
CD19
CD22
CD10
CD33
T lineage
CD34
CD7
CD3
TdT
CYTOGENETICS IN ACUTE
LEUKAEMIA
• Translocations, deletions, inversions etc
• Diagnosis
• Prognosis
• Minimal Residual Disease (MRD)
Investigations
1. Full blood count
reduced haemoglobin
normochromic,
normocytic anaemia,
WBC
<1.0x109/l to >200x109/l,
neutropenia and f blast
cells
Thrombocytopenia
<10x109/l).
Investigations
Acute lymphoblastic
leukemia
Acute myeloid leukemia
Investigations
ALL(Lymphoblast)
– Blast size :small
– Cytoplasm: Scant
– Chromatin: Dense
– Nucleoli :Indistinct
– Auer-rods: Never
present
AML (Myeloblast)
– Large
– Moderate
– Fine, Lacy
– Prominent
– Present in 50%
Investigations
2. Bone marrow
aspiration and
trephine biopsy
 confirm acute
leukaemia
(blast > 30%)
usually
hypercellular
Investigations
3.Cytochemical
staining
a) Peroxidase :* negative ALL
* positive AML
Positive for myeloblast
Sudan black

Sudan black stains phospholipids, neutral fats and sterols
found in primary and secondary granules of granulocytic cells
and to a lesser extent in monocytic lysosomes. Rare positives
occur in lymphoid cells
Nonspecific Esterase

Nonspecific esterase – is used to identify monocytic cells
which are diffusely positive. T lymphocytes may have focal
staining
Leukocyte Alkaline phosphatase

Leukocyte alkaline phosphatase – is located in the tertiary
granules of segmented neutrophils, bands and
metamyelocytes. The LAP score is determined by counting
100 mature neutrophils and bands. Each cell is graded from 0
to 5. The total LAP score is calculated by adding up the
scores for each cell.
Investigations
b)Periodic acid schiff
*Positive ALL
(block)
* Negative AML
Block positive in ALL
Investigations
c) Acid phosphatase :
focal positive
(T-ALL)
Investigations
4.Immunophenotyping
 identify antigens present on the blast cells
 determine whether the leukaemia is
lymphoid or myeloid(especially important
when cytochemical markers are negative or
equivocal. E.g : AML-M0)
 differentiate T-ALL and B-ALL
Monoclonal antibodies(McAb) are group
based on antigen on the leucocytes and
are recognised under a cluster of
differentiation(CD).
Certain antigens have prognostic
significance
Rare cases of biphenotypic where both
myeloid and lymphoid antigen are
expressed
Able to identify the subtype of leukemia.
E.g : AML-M7 has a specific surface
marker of CD 61 etc
Monoclonal antibodies(McAb) are recognised under a
cluster of differentiation(CD).
MONOCLONAL ANTIBODIES USED FOR
CHARACTERISATION OF ALL AND AML.
Monoclonal antibodies
AML :
CD13, CD33
ALL : B-ALL
CD10, CD 19, CD22
T-ALL
CD3, CD7
Investigations
5.Cytogenetics and molecular studies
detect abnormalities within the leukaemic
clone
diagnostic or prognostic value
E.g : the Philadelphia chromosome : the
product of a translocation between
chromosomes 9 and 22
confers a very poor prognosis in ALL
Investigations
COMMON CHROMOSOME ABNORMALITIES
ASSOCIATED WITH ACUTE LEUKEMIA
t(8;21)
AML with maturation (M2)
t(15;17) AML-M3(APML)
Inv 16
AML-M4
t(9;22) Chronic granulocytic leukemia
t(8;14) B-ALL
Others Invx
6.Biochemical screening
leucocyte count very high - renal impairment
and hyperuricaemia
7.Chest radiography
 mediastinal mass - present in up to 70% of
patients with T -ALL
In childhood ALL bone lesions may also
seen.
Others Invx
8.Lumbar puncture
initial staging inv. to detect leukaemic cells
in the cerebrospinal fluid, indicating
involvement of the CNS
Done in acute lymphoblastic leukemia
Mechanisms of intervention of leukemic cells in the
activation of blood coagulation
GENETICS
Leukemic mutations interfere with transcription
factor functions, abrogate cell differentiation,
and support proliferation. As a consequence,
the blood is flooded with immature, nonfunctional cell types.
Molecular Pathology of AML
Abnormal cell proliferation
– FLT3 mutations
– Ras mutations
– Others: c-KIT mutations
Block in differentiation
– CBF AML (t(8;21) and inv(16))
– PML-RARα (t(15;17))
– MLL translocations (11q23)
– Hox gene translocations
– C/EBPα mutations
Suppression of apoptosis
– Bcl-2 over-expression
Capacity for indefinite self-renewal
ACUTE LEUKAEMIA
EXAMPLES of SOME
CYTOGENETIC and MOLECULAR
ABNORMALITIES
Proliferation: FLT3
A receptor tyrosine kinase
expressed in 70 – 100% of AML
cases.
Activating mutations in FLT3 are
seen in ~30% of AML cases.
– Tandem duplication of the
juxtamembrane region.
– Point mutation within the
activation loop of the kinase
domain.
Activation of FLT3 leads to
deregulated proliferation of AML
cells.
Proliferation: Ras
Small guanine nucleotide binding proteins that play a
role in several cell signaling pathways.
Activating point mutations seen in ~25% of AML cases.
– N-Ras: 10 – 25%
– K-Ras: 5 – 15%
– H-Ras: Rare
Activation of Ras leads to deregulated cell proliferation.
Differentiation: t(15,17)and the
PML/RARa gene
Acute Promyelocytic Leukaemia
- gene: retinoic acid receptor (RARa) gene
fused with PML gene (PML/RARa gene)
This fusion PML-RAR protein is responsible for
preventing immature myeloid cells from
differentiating into more mature cells.
t(8;21) - [AML1-ETO]
The AML1 gene encodes the DNA-binding subunit
of the AML1/CBFb core binding factor transcription
complex, whereas ETO encodes the mammalian
homologue of the Drosophila protein Nervy.
AML1 and ETO are both involved in transcriptional
regulation of genes in hematopoietic precursor
cells.
AML1-ETO fusion protein represses genes whose
transcription is normally activated by AML/CBFb.
The AML1-CBFß Transcription
Factor
In normal cells, heterodimeric
AML1-CBFß transcriptionfactor complex binds to the
DNA sequence TGTGGT in
the transcriptional
regulatory region of AML1regulated target genes and
activates transcription
through the recruitment of
coactivators.
The AML1-CBFß Transcription Factor
In AML cells with the t(8;21)
translocation, the N-terminal
part of AML1 fuses with the
C-terminal portion of ETO.
The resultant chimeric
protein continues to interact
with CBFß and to bind to the
core enhancer sequence;
however, ETO recruits a
nuclear corepressor
complex and results in the
dominant repression of
AML1-regulated target
genes.
The AML1-CBFß Transcription Factor
Similarly, the CBFß-MYH11
chimeric protein encoded
by the inv(16) mutation
continues to interact with
AML1; however, instead of
allowing AML1 to interact
with DNA, this chimeric
protein recruits AML1 into
functionally inactive
complexes in the
cytoplasm.
In summary
Differentiation: Core Binding Factor-β
CBFβ is located at 16q22.
Inv(16)(p13q22) and t(16;16)(p13;q22): CBFβMYH11 fusion gene that functions as a
dominant negative inhibitor of CBF.
The result is the suppression of transcription of
genes crucial for hematopoiesis / differentiation.
Differentiation: the Mixed Lineage
Leukemia (MLL) Gene
Mixed-lineage leukemia (MLL) fusion proteins.
There are more than 40 proteins that have been
found fused to MLL in leukemia patients, and
different ones can cause leukemia by different
mechanisms.
When the transcription factor MLL functions as it
should, without a fusion partner, it binds to and
controls the expression of Hox genes, which in turn
control cell growth and maturation.
Differentiation: the Mixed Lineage
Leukemia (MLL) Gene
MLL is a protein that
regulates Hox gene
expression.
Hox family members play
critical roles in transcription
of genes involved in
hematopoiesis.
Normal MLL requires
cleavage by taspase into
its mature form →
dysregulated Hox gene
expression → block in
differentiation.
t (12,21)
- most common in childhood ALL
- gene: AML1 and TEL
- “extremely” high long-term survival
Pathogenesis: Summary
Strategies that interfere with over-activation of
signaling pathways and the block in cellular
differentiation have the potential to improve patient
outcomes in AML.
Approach to acute leukaemia
• In all, but the elderly or medically unfit, intensive initial
treatment with combinations of chemotherapeutic
drugs is indicated
• The aim is to rapidly clear leukaemic cells from the
marrow and to restore normal bone marrow function,
ie achieve REMISSION
• CURE is the goal
Principles of treatment
combination chemotherapy
– first goal is complete remission
– further Rx to prevent relapse
supportive medical care
– transfusions, antibiotics, nutrition
psychosocial support
– patient and family
Response to treatment
• Childhood ALL:
>98% remission
80% cure
• Adult AML and ALL: 60-75% remission
25-35% cure
Prognosis in AML
parameters
Favorable
unfavorable
Cytogentics
T(15;17).
T(8;21).
Inv(16).
Deletion of chromosome5or7.
11q23
T(6;9)
Abn(3q)complex rearrangments
<5% blasts after first course
BM response to
remission induction
>20% blasts after first course.
age
>60yrs
<60yrs
Current Research
Inhibition of the products of genetic mutations found in AML cells
Inhibition of proteins that cause chemotherapy resistance
Use of antibody therapy against AML cells
Use of new or existing drugs given in different doses and
schedules
Techniques to make stem cell transplantation safer, easier, and
more effective
Evaluation of drugs called hypomethylating therapy