Introduction and Basics Clonal hematopoietic stem cell disorder lt 2 of all paediatric leukemias Consistent chromosomal abnormality Philadelphia chromosome translocation t922 q34q112 ID: 930043
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Slide1
25/4/2017
CML : MOLECULAR PATHOLOGY AND CLINICAL MANIFESTATIONS
Slide2Introduction and Basics
Clonal
hematopoietic stem cell disorder
< 2% of all paediatric
leukemias
Consistent chromosomal abnormality :Philadelphia chromosome translocation t(9;22) (q34;q11.2)
Incidence :
1-4 yrs : 0.2%
5-9 yrs: 2.2%
10-14 yrs: 3.7%
15-19 yrs: 8.3%
Slide3Slide4Historical background
First leukemia to be recognized as a disease entity
1844 – Donne – hematological changes
1845 – Bennett,
Craigie
, Virchow – clinical features & autopsy findings
Virchow classified leukemia as
splenic
and lymphatic
1879- Neumann – Leukemic cells originated in BM
1889 –
Ebstein
– classified as acute / chronic
1960 – Ph chromosome – 1
st
chr
abnormality associated with malignancy (
Nowell
& Hungerford)
Slide5Historical background
Treatment history:
1865 - Potassium arsenate (Fowler’s solution) –
Lissauer
– limited & temporary improvement
1902 – radiotherapy – Pusey – more predictable and better results – less toxic
1953 –
Busulfan
introduced
Later –
hydroxyurea
, interferon-alpha,
Cytarabine
,
but none produced sustained remissions
1970 –
allo
HSCT
Later –
Imatinib
- durable remission, excellent quality of life
Slide6Clinical presentations
Leading symptoms
Asthenia (45%–60%),
Splenic
discomfort (20%–30%)
Weight loss (15%–20%)
Bleeding (10%)
30 percent of patient are asymptomatic at the time of diagnosis
Median WBC count:240 x10
9
/L (range - 10–720 X10
9
/L) – higher than adult CML
Anemia or
thrombocytosis
: 60%
Splenomegaly
(60%–70%) – Higher TLC
Slide7CML-BP
discriminating CML-BP & Ph + acute leukemia may be impossible
No ethnic/genetic predisposition
Ionizing radiation a risk factor but have not been demonstrated to be causal in children
Slide8Less frequent symptoms:
Night sweats
Heat intolerance- mimicking hyperthyroidism
Symptoms of
leukostasis
(tinnitus,
stupor,priapism
)
Splenic
infartion
(left upper-quadrant and left shoulder pain)
Urticaria
(result of histamine release)
Slide9NATURAL HISTORY
CML-CP : Approximately 95
%
CML-AP 5%
CML-BC
Slide10Chronic phase
Median duration
5–6 years
Accelerated phase
Median duration
6–9 months
Blast crisis
Median survival
3–6 months
Advanced phases
Faderl
S, et al.
Ann Intern Med
. 1999;131:207-219.
Pasternak G, et al.
J Cancer Res
Clin
Oncol
. 1998;124:643-660.
Clinical
Course: Phases of Untreated CML
p53, Rb, p16, t(3;21),
t(8;21), t(7;11)
Slide11Biology of CML
CML-BP :
Myeloid (60%–70%)
Lymphoid (20%–30%),
Mixed-lineage phenotype
CML-BP without a diagnosed preceding CML-CP.
Slide12Criteria for phases in CML
CML-CP (95%)
CML-AP
CML-BC
Must meet all of the following criteria
Must meet 1 or more of the following criteria
Must meet 1 or more of the following criteria
t(9;22) or the
Bcr-Abl
fusion gene
Blasts 10%-19% in peripheral blood/bone Marrow
Blasts 20% in peripheral blood/bone marrow
Bone marrow blasts 10%
Peripheral
basophils
≥ 20%
Extramedullary
blast proliferation
Does not meet any criteria for AP/BC
Persistent thrombocytopenia/
thrombocytosis
unrelated to Rx
Large foci or clusters of blasts in BM biopsy
↑↑ spleen size and WBC count unresponsive to Rx
Cytogenetic evidence of
clonal
evolution
Megakaryocyte
proliferation/marked
reticulin
or collagen fibrosis, and/or severe granulocytic dysplasia
Slide13Pathology
Pediatric
CML & adult CML : Same molecular feature
balanced translocation t(9;22)(q34;q11)
fusion gene
BCR-ABL
BCR-ABL encodes a constitutively active tyrosine
kinase
Either as a 210
kDa
in CML or 190
kDa
in B-cell lineage ALL
Slide14Ph chromosome is formed by a reciprocal translocation between the long arms of chromosomes 9 and 22, causing the juxtaposition of the BCR (breakpoint cluster region) and ABL1 (Abelson) genes
BCR-ABL1 fusion gene: 5′ end of the BCR gene and the 3′ end of the ABL1 gene
Slide15Melo.
Blood
. 1996;88:2375.
Pasternak et al.
J Cancer Res Clin Oncol
. 1998;124:643.
Ph
Chromosome and the
bcr-abl
Gene:
t(9;22
)
Translocation
FUSION PROTEIN WITH CONSTITUTIVE
TYROSINE KINASE ACTIVITY
bcr-abl
bcr
Philadelphia Chromosome (or 22q-)
Chromosome 9 q+
abl
Chromosome 9
Chromosome 22
Slide16Reciprocal translocation a segment of
ABL gene
(9q34) is moved into one of at least 3 well characterized breakpoints of the
BCR gene in 22q112,3.
Results
in two fusion genes
BCR-ABL and ABL-BCR. Of this,
the
ABL –BCR has no identified role in pathogenesis
of CML
Slide17Three
different
break points in BCR
Exon
(e1
),
Exons
(b2/b3) and
Exons
(e19) to ABL4.
Breakpoints in
ABL
Upstream
in ABL
exon2 (a2)
Downstream of exon2 (a3).
Resulting tyrosine
kinase
proteins
p185/190 (minor
transcript or ‘m’) p210 (Major transcript or ‘M’)P230 (u-BCR: Ph+ Chronic neutrophilic Leukemia){Note: BCR exons
12-16 (originally known as exons b1 – b5)}BCR-ABL fusion gene
Constitutively active tyrosine kinase (oncoprotein)
Slide18Slide19Slide20Slide21Slide22Chimeric
BCR-ABL fusion protein
constitutively activated tyrosine
kinase
(TK)
Autophosphorylation
downstream pathways
proliferative signal responsible for regulation of growth and replication
Creates a cytokine-independent cell cycle with aberrant apoptosis
Slide23Normal Bcr-Abl
Signaling*
The
kinase
domain activates a substrate protein,
eg
, PI3
kinase
, by
phosphorylation
This activated substrate initiates a signaling cascade culminating in cell proliferation and survival
P
P
P
ADP
P
P
P
P
P
ATP
SIGNALING
Bcr-Abl
Substrate
Effector
ADP = adenosine diphosphate; ATP = adenosine triphosphate;
P = phosphate.
Savage and Antman.
N Engl J Med.
2002;346:683
Scheijen and Griffin.
Oncogene.
2002;21:3314.
Slide24Important Pathways Affected by BCR-ABL1 Activity
JAK/STAT
PI3K/AKT and
Autophagy
Ras
/MEK
Src
Kinases
Crkl
Long
Noncoding
(
lnc
) RNA-BGL3
Slide25Apoptosis Deregulation:
In addition to promoting cell proliferation, BCR-ABL1 can disrupt cell death
leading to defective apoptosis
Slide26Consequences of breakpoint
Patients with e14a2 transcript or both the e14 and e13a2 transcripts
higher platelet count approximately 1.5 times higher than that in the e13a2 group
(Balatzenko et al. 2011 ; Hanfstein et al. 2014 ;
Jain et al. 2015 )
Patients with e14a2 transcript: faster rate of achieving a deeper response (MMR, i.e., a 3-log reduction)
Slide27CML Stem Cells
Quiescent
leukaemic
stem cells (LSCs) within the CD34+ population are resistant to TKIs
(Bhatia et al. 2003 ; Copland et al. 2006 ; Jorgensen et al. 2007 )
Responsible for relapse in approximately half of all patients eligible for therapy cessation
(Mahon et al. 2010 )
Slide28TK inhibition reduced LSC proliferation, but does kill quiescent LSCs
(Graham et al. 2002 )
LSCs were also insensitive to more potent second-generation TKIs, even though the BCR-ABL1
kinase
activity was silenced
(Copland et al. 2006 ; Jorgensen et al. 2007 )
Slide29LSCs may not require BCR-ABL1 for survival, and/or rely on non-
kinase
activity of BCR-ABL1, and/or prefer moderate
kinase
activity
Several pathways have been shown to play key roles in stem cell biology, and targeting them could lead to a promising strategy to eliminate the LSC in CML
Slide30Biology of blast crisis
Progenitor cells gain self-renewal capacity, differentiation arrest and survival properties that lead to their uncontrolled proliferation
(Jamieson et al. 2004 )
BC progenitors exhibit more stem cell-like characteristics compared to CP progenitors
Slide31Biology of blast crisis
Partially attributed to increased β-
catenin
activity
Genomic and genetic instability is another feature of advanced disease
(
Perrotti
et al. 2010 ;
Skorski
2012 )
Extrachromosomal
abnormalities are observed in approximately 80 % of BC patients (e.g. Ph duplication,
trisomy
8 or 19, loss of 17p)
(Johansson et al. 2002 )
Slide32Additional chromosome aberrations in
CML-CP at diagnosis
Incidence in CML –CP at diagnosis is 3-5%
Most common additional aberrations
Loss of Y chromosome
Gain of chromosome (
trisomy
) 8
Duplication of Ph
i
(17q)
Except loss of Y, all are considered as major route abnormalities and are associated with decreased survival and signify progression to accelerated phase and blast crisis : WARNING FEATURE
Slide33Cytogenetic evolution during the
Accelerated and
Blastic
Phases in CML
Additional Ph (30-32%)
Trisomy
8 (28-33%)
i
(17q) (20%)
Trisomy
of chromosome 19(12%-19%)
Trisomy
21 ( 7- 9%)
Loss of the Y chromosome (8% in males)
Loss of chromosome 7 (5%) in 60-80% of cases
May occur individually or in combination
Clonal
evolution which seems to play an important role in
Imatinib
Mesylate
resistance
Slide34CML – Peripheral Blood Findings
Peripheral smear can only give a presumptive
diagnosis of CML [you need to confirm the t(9;22)]:
1)
leukocytosis
with a ‘left shift’
2)
normocytic
anemia
3)
thrombocytosis
in 50% of pts
4) absolute
eosinophilia
with a normal % of Eos.
5) absolute and relative increase in
basophils
6) LAP score is low (not frequently employed)
Slide35Laboratory
features
BM: Marrow
is
hypercellular
granulocytic
hyperplasia
Reticulin
fibrosis
Cytogenetic
test- presence of the Ph chromosome
Molecular test – presence of the BCR-ABL fusion gene
Slide36Diagnostic Tests in CML
Karyotyping
in CML
1) Allows for the diagnosis of CML
2) Requires a bone marrow aspirate for
optimal metaphases
3) Allows for evaluation of
clonal
evolution as
well as additional chromosomal abnormalities
in the non-Ph
+
clones
4) Occasional cryptic and complex
karyotypes
can result in the missed identification of the
t(9;22)
Demonstrating the presence of the t(9;22) or its gene product is
absolutely essential in diagnosing a patient with CML
Slide37Diagnostic Test in CML
Fluorescence in-situ hybridization
(FISH) in CML
:
1)
Allows for the diagnosis of CML
2) Does not require a bone marrow aspirate for
optimal results
3) Allows for the identification of potential
duplications of the Ph chromosome
4) Allows for the identification of the loss of the
der
(9)
chromsome
5) Allows for the identification of cryptic
translocations involving
Bcr-Abl
Bcr- Ch 22
Abl – Ch 9
Bcr-Abl Fusion
Slide38Diagnostic Test in CML
Bcr-Abl
Bcr
Abl
cDNA
Quantitative RT-PCR
for
Bcr-Abl
in CML
1) Allows for the diagnosis of CML
2) Does not require a bone marrow aspirate for
optimal results
3) Can quantify the amount of disease
4) Allows for the identification of cryptic
translocations involving
Bcr-Abl
5) Many primers sets only detect the p190 and/or
the p210 translocation and may miss the p230
or alternative translocations
Slide39Disease Diagnosis and
Monitoring in CML
Test
Target
Tissue
Sensitivity (%)*
Use
Cytogenetics
Ph chromosome
BM
1
-
10
▪
Confirm diagnosis of CML
▪
Evaluate karyotypic
abnormalities other than Ph
chromosome (ie, clonal
evolution)
FISH
Juxtaposition of
bcr
and
abl
PB/BM
0.5
-
5
▪
Confirm diagnosis of CML
▪
Routine monitoring of
cytogenetic response in
clinically stable patients
▪
Routine measurement of
MRD
RT-PCR
bcr-abl
mRNA
PB/BM
0.0001
-
0.001
▪
Routine measurement of
MRD
▪
Determine the breakpoints of
the fusion genes
*Number of leukemic cells detectable per 100 cells.
BM = bone marrow; FISH = fluorescence in situ hybridization; PB = peripheral blood;
MRD = minimal residual disease; RT-PCR = reverse transcriptase polymerase chain reaction.
Wang et al.
Genes Chromosomes Cancer
. 2001;32:97
Slide4015 years/Male
Presented with
Weakness and decreased appetite for 8 weeks
Liver 2 cm Spleen 15 cm BCM
Slide41Slide42Slide43Slide44Slide45Slide46Thank you