25/4/2017 CML : MOLECULAR PATHOLOGY AND CLINICAL MANIFESTATIONS - PowerPoint Presentation

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25/4/2017

CML : MOLECULAR PATHOLOGY AND CLINICAL MANIFESTATIONS

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Introduction 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%

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Historical 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)

Slide5

Historical 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

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Clinical 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

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CML-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

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Less 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)

Slide9

NATURAL HISTORY

CML-CP : Approximately 95

%

CML-AP 5%

CML-BC

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Chronic 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)

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Biology of CML

CML-BP :

Myeloid (60%–70%)

Lymphoid (20%–30%),

Mixed-lineage phenotype

CML-BP without a diagnosed preceding CML-CP.

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Criteria 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

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Pathology

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

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Ph 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

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Melo.

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

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Reciprocal 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

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Three

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)

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Chimeric

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

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Normal 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.

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Important Pathways Affected by BCR-ABL1 Activity

JAK/STAT

PI3K/AKT and

Autophagy

Ras

/MEK

Src

Kinases

Crkl

Long

Noncoding

(

lnc

) RNA-BGL3

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Apoptosis Deregulation:

In addition to promoting cell proliferation, BCR-ABL1 can disrupt cell death

 leading to defective apoptosis

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Consequences 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)

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CML 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 )

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TK 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 )

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LSCs 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

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Biology 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

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Biology 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 )

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Additional 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

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Cytogenetic 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

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CML – 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)

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Laboratory

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

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Diagnostic 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

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Diagnostic 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

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Diagnostic 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

Slide39

Disease 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

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15 years/Male

Presented with

Weakness and decreased appetite for 8 weeks

Liver 2 cm Spleen 15 cm BCM

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Thank you