MOLECULAR GENETICS OF MYELOPROLIFERATIVE

DISORDERDSDr. A. Arun Kumar

Moderator: Dr. Mili Jain

General Pathogenesis of Myeloid neoplasms

Class I MutationsPROMOTE PROLIFERATION Class II Mutations

BLOCKS DIFFERENTIATION

BCR-ABLFLT3JAK 2 etc…

PML/RARAC/EBPA etc

PROLIFERATIVE DISORDER

ACUTE MYELOID NEOPLASMS

Genetic abnormality Disease FrequencyBCR-ABL CML 99%

JAK2V617F

PV 95%ET 60%

PMF 60%MPN-U 20%RARS-T 50%

JAK2 EXON 12 PV 2%MPLW515L/K PMF 8%

  ET 8%

 PDGFRA

Myeloid neoplasms with eosinophilia Unknown

Mast cell disease  

PDGFRB Myeloid neoplasms with eosinophilia Unknown

FGFR Myeloid neoplasms with eosinophilia Unknown

Involving KIT (D816V - most frequent) Mast cell disease Unknown

General scheme for tyrosine kinases

Kinase domain

Upregulating domain [SH2]

Inhibitory domain[SH3]

“The findings suggest a causal relationship between the chromosome abnormality observed and chronic granulocytic leukemia.”Peter Nowell

1960 Nowell and Hungerford find that

one copy of chromosome 22 in

CML patients is always extremely short

BCR-ABL

The Philadelphia chromosome - originally in Philadelphia.

The first genetic abnormality to be associated with a human cancer.

The result of a balanced translocation between chromosomes 9 and 22.

Derivative chromosome 22 is significantly smaller Ph chromosome is present in all hematopoietic lineage

cells from patients with CML. Ph chromosome is acquired and NOT inherited

through the germline.

Melo. Blood. 1996;88:2375. Pasternak et al. J Cancer Res Clin Oncol. 1998;124:643.

Breakpoints in BCR: Major (M) – exons 12 to 16 [b1,b2,b3,b4 & b5]

Minor (m) – exons 1 & 2 [e1, e2, e11 & e21]

Mu (μ) – exons 19& 20 [e19 & e20]

Breakpoint in ABL a2

b3a2 or b2a2 p210[Adult and childhood CML]

e1a2 p190 [ALL]

e19a2 p230 [neutrophilic CML]

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

PP P

ADP P

P

PP PATP

SIGNALING

Bcr-Abl

Substrate

Effector

Savage and Antman. N Engl J Med. 2002;346:683Scheijen and Griffin. Oncogene. 2002;21:3314.

Imatinib Mesylate: [Gleevac/STI 571] Mechanism of Action* Imatinib mesylate

occupies the ATP binding pocket of the Abl kinase domain

This prevents substrate phosphorylation and signaling

A lack of signaling inhibits proliferation and survival

P

PP PATP

SIGNALING

Imatinib mesylate

Bcr-Abl

Savage and Antman. N Engl J Med. 2002;346:683.

Molecular Cell BiologyLodish et al. Fig. 24.1

CML arises in a stem cell that is a granulocyte precursor ???

Leukaemogenesis

Ph chromosome is found on myeloid, monocytic, erythroid, megakaryocytic, B-cells and sometimes T-cells

Molecular Cell BiologyLodish et al. Fig. 24.1

CML arises in a stem cell that is a granulocyte precursor ???

BCR-Abl

Cytoskeleton/adhesion defects

SG2

M

1GG0

Apoptosis

Stem cell turnover

Proliferation & differentiation

BCR-Abl affects multiple cell functions

Adapted from Jörgensen, 2001. Hem. Onc.

Diagnosing Chronic Myeloid Leukemia

DEMONSTRATION OF PHILADELPHIA CHROMOSOME

Diagnostic Considerations in Chronic Myeloid Leukemia

Karyotyping in CML

1) Allows for the diagnosis of CML2) Requires a bone marrow aspirate for optimal metaphases3) Allows for evaluation of clonal evolution as well as additional chromosomal abnormalities in the non-Ph+ clones4) 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

Source Undetermined

FISH in CML

Red → Bcr probeGreen → Abl Probe

Yellow → fusion of Bcr and Abl

Ch 9 Ch 22

Bcr- Ch 22

Abl – Ch 9

Bcr-Abl Fusion

Source Undetermined

Fluorescence in-situ hybridization(FISH) in CML:

1) Allows for the diagnosis of CML2) Does not require a bone marrow aspirate for optimal results3) Allows for the identification of potential duplications of the Ph chromosome 4) Allows for the identification of the loss of the der (9) chromsome5) Allows for the identification of cryptic translocations involving Bcr-Abl

Diagnostic Considerations in Chronic Myeloid Leukemia

Bcr-Abl

Bcr

Abl

cDNA

Quantitative RT-PCR for Bcr-Abl in CML

1) Allows for the diagnosis of CML2) Does not require a bone marrow aspirate for optimal results3) Can quantify the amount of disease4) Allows for the identification of cryptic translocations involving Bcr-Abl5) Many primers sets only detect the p190 and/or the p210 translocation and may miss the p230 or alternative translocations

Source Undetermined

Disease Diagnosis and Monitoring in CML

Test Target Tissue Sensitivity (%)*

Use

Cytogenetics Ph chromosome BM usually in Heparin

1-10 Confirm diagnosis of CML Evaluate karyotypic abnormalities other than Ph chromosome (ie, clonal evolution)

FISH Juxtaposition of bcr and abl

PB/BM usually in Heparin

0.5-5 Confirm diagnosis of CML Routine monitoring of cytogenetic response in clinically stable patientsRoutine measurement of MRD

RT-PCR bcr-abl mRNA PB/BM in EDTA 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

Role of BCR-ABL in therapeutic monitoring

Complete Hematological Response

LEVEL OF CYTOGENETIC RESPONSE CONVENTIONAL CYTOGENETIC ANALYSIS

Minimal Cytogenetic Response 66-95% Ph + metaphases

Minor Cytogenetic Response 36-65% Ph + metaphases

Partial Cytogenetic Response 1-35% Ph + metaphases

Complete Cytogenetic Response 0% Ph + metaphases

LEVEL OF MOLECULAR RESPONSE RT – PCR ANALYSIS

Major Molecular Response >= 3-log reduction of BCR-ABL mRNA

Complete Molecular Response Negative by RT-PCR

Therapeutic significance of BCR-ABL

Imatinib – selective Abl specific tyrosine kinase inhibitor. Binds to only the inactive conformation of Abl kinase domain

Nilotinib – selective abl inhibitor. Binds to only the inactive conformation of Abl kinase domain

Dasatinib – Src/ABL inhibitor. Binds to both active and inactive conformation of Abl kinase domain

“Pseudo kinase” domain -autoinhibition of kinase

JAK 2

V617F

CytokineReceptor Binding

Kinase Domain

JH1JH2JH3JH4JH5JH6JH7

*Wernig et al Blood 2006 Lacout et al Blood 2006 Zaleskas et al. PLoS One 2006 Bumm et al Can Res 2006

JAK2 mutations

FERM SH2 JH2 Kinase

exon 14 V617F

95% PV

50% ET

50% IMF

neg

exon 12 mutations

V617F-neg PV variant

exon 16 R683

Acute Lymphoblastic Leukaemia

Where is the JAK2 gene located?

Cytogenetic location: 9p24

The JAK2 gene is located on the short (p) arm of chromosome 9 at position 24.

Mutations in JAK2  

polycythemia vera

essential thrombocythemia

Myelofibrosis

Other MPDs & MDS/MPN

Mutation:

GOF mutation

GT somatic mutation in exon 14 V617F

valine to phenylalanine at the 617 position,

The JAK2 gene synthesize protein that promotes the growth and division (proliferation) of cells.

This protein is part of a signaling pathway called the JAK/STAT pathway, which transmits chemical signals from outside the cell to the cell's nucleus.

JAK 2 Mutations

Polycythemia Vera

Essential Thrombocytosis

Primary Myelofibrosis

Myelodysplastic syndromes

AML

MDS/MPN aCML

CMML

RARS-T

????????? Why is the same mutation occurring in the same

gene (V617F in JAK 2 gene) cause PV in some patients and ET or PMF in others ????

Why do some ET patients progress to PMF while others progress to PV ????

Janus kinase 2 has been shown to interact with:

DNAJA3 EGFR EPOR TPOR TNFRSF1A VAV1

Grb2 GHR IRS1 IL12RB2 STAT5B STAM

IL5RA PIK3R1 PPP2R4 PTK2

PTPN6 PRMT5

SOCS3 STAT5A

SOCS1 TEC  

YES1 FYN

Heterozygous JAK2V617F

9p LOH

ET

PV

JAK2V617F knockin mouse – homozygosity causes phenotypic switch from ET to PV

Li et al Blood 2010 Juan Li, David Kent unpublished

Pla

tele

ts (

x10

3 )/µ

l

Ha

em

ato

crit

(%)

WT Het Hom WT Het Hom

Haematocrit Platelets

Inter relationship among Ph - MPNs

ET PV PMF

Beer et al Blood 2011

Nomenclature of “prefibrotic PMF” is flawed

“True ET”

PV PMF

Prefibrotic PMF?

Nomenclature of “prefibrotic PMF” is flawed

ET PV PMF

Prefibrotic PMF?

Nomenclature of “prefibrotic PMF” is flawed

ET PV PMF

Prepolycythaemic PV?

JAK2V617F negative MPN• JAK2V617F-negative PV

- JAK2 exon 12 mutations- loss of function mutations in LNK, negative regulator of

JAK2*

• JAK2V617F-negative ET/PMF- MPL mutations in 10%- LNK mutations in <5%

• Somatic mutations have not been identified in few MPN patients

* Oh et al Blood 2010

MPL mutations In 2006, 2 novel MPL somatic mutations (MPL W515L and MPL W515K) have

been discovered in 5% and 1%, respectively, of JAK2 V617F–negative PMFs. 

Cytogenetic Location: 1p34

Replacement of the amino acid tryptophan at position 515 with another amino acid, most commonly leucine. [MPLW515L]

Amino acid changes at position 505 or 515 result in a thrombopoietin receptor protein that is constantly turned on without a stimulation

Disorders with MPL mutations Essential Thrombocythemia [MPLW515L]

Familial Essential Thrombocythemia [MPLW505]

Primary Myelofibrosis

Congenital Amegakaryocytic Thrombocytopenia[CAMT]

Kit/SCFR pathwayThe KIT protein/SCFR cell membrane of certain cell types

stem cell factor, attaches (binds) to it.

This binding turns on (activates) the KIT protein

activates tyrosine kinases

Phosphorylation

proliferation.

C- KIT Germ cells, hematopoietic stem cells, mast cells, Interstitial cells of Cajal (ICCs), melanocytes.

CD117, C-Kit, Mast/Stem cell growth factor receptor (SCFR)

Proto-oncogene c-Kit, Tyrosine-protein kinase Kit.

Cytogenetic Location: 4q12

Mutation: codon 816 Aspartate to Valine [D816V]

DISEASES : Mast cell diseases

Few cases of AML, NK/T cell lymphoma

GIST

Piebaldism

SIGNIFICANCE:

C-KIT mutated mastocytosis patints respond to IMATINIB

PDGFRA

Associated in myeloid neoplasms with eosinophilia Separate category in WHO 2008 Classification of Myeloid Neoplasms

[Cat 5: Myeloid neoplasms with Eosinophilia & abnormalities of PDGFRA, PDGFRB and FGFR1]

Deletion fusion of two genes FIP1L1 – PDGFRA Chronic eosinophilic leukemia

SM with eosinophilia

PDGFRA C-Kit

Chromosome 4q Chromosome 4q

Tyrosine kinase Tyrosine kinase

Systemic Mstocytosis Systemic Mstocytosis

GIST GIST

OTHER MUTATIONSMUTATION MYELOID DISORDERS

PDGFRB Myeloid neoplasms with EosinophiliaCMML with EosinophiliaACML

RAS CMMLJMML

TET 2 MPN progression to AMLCMMLFew cases of AML & MDS

ASXL1 MPN progression to AML

http://ghr.nlm.nih.gov/

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