internal medicine, yr 5 basic concept of the treatment of malignancies dank magdolna ... anemia,...

Csaba Bödör

Semmelweis University1st Department of Pathology and Experimental Cancer Research

Basic molecular aspects of hematological diseases

from bench to bedside with case presentations

October 14, 2013, Internal Medicine, 5th year [email protected]

Date Title of the lecture Lecturer

Sept. 9. Classification of malignancies, pathological and

molecular-pathological aspects

Kulka Janina

Sept. 16. The basic concept of the treatment of malignancies Dank Magdolna

Sept. 23. Diagnose and treatment availability of breast cancer Dank Magdolna

Sept. 30. GI cancer: Gastric, pancreatic and liver malignancies Orosz

Zsuzsanna

Oct. 7. GI cancer: Colorectal malignancies Szentmártoni

Gyöngyvér

Oct. 14. Basic molecular aspects of hematological diseases –

from bench to bedside with case presentations

Bödör Csaba

I Pathol

Oct. 21. Approach to the patient with anaemia: iron deficiency,

megaloblastic, hemolytic, aplastic anaemias

Demeter Judit

Oct. 28. Indolent and high grade non-Hodgkin lymphomas,

Hodgkin disease

Demeter Judit

Nov. 4. Myelodyplasia, acute leukemias Nagy Zsolt

Nov. 11. Chronic myeloproliferative neoplasms (CML, PV, ET,

PMF)

Demeter Judit

Nov. 18. Thrombophilia, hemophilia Domjan Gyula

Nov. 25. Multiple myeloma and other plasma cell diseases,

plasmapheresis

Nagy Zsolt

Dec. 2. Immunhematological disorders Demeter Judit

Dec. 9. Stem cell transplantation Nagy Zsolt

Internal Medicine, Yr 5

Introduction

Complex diagnostics of hematological malignancies

Morphology

FISH

CytogeneticsFlow cytometry

Q-PCRSequencing

Introduction

What is the rationale of molecular analyses in hem. malignancies ?

Diagnostic markers (i.e. BCR-ABL1/CML)

Prognostic markers (i.e. FLT3-NPM1-CEBPA/AML)

Follow up - MRD detection (i.e. IgH clonality analysis)

Therapeutic targets (i.e. BCR-ABL1/CML; CD20/B-cell lymphomas)

Basic Concepts

”From bench to bedside”

From ”One size fits all” model to ”Personalized Treatment”

responders

non-responders

Genetic abnormalities in hematological malignancies

The success of TKIs in Chronic Myeloid Leukemia (CML)

The JAK2 story in Myeloproliferative Neoplasms (NPM)

The changing genetic landscape of AML

Clonality assessment and MRD detection in lymphoid malignancies

Gene expression and mutation profiling in diffuse large B-cell lymphoma

The emerging role of epigenetic deregulation in B-cell lymphomas

Chronic Myeloid Leukemia (CML)

The (historical) course of the disease

DeathBlast

crisisAccelerated

phase

Chronic

phase

3-5 years 3-6 months 3 months


Paradigm shift in oncology > targeted therapy

Philadelphia chromosome

t(9;22) translocation

BCR-ABLfusion gene

1960 1973 1985





BCR-ABLfusion gene

1960 1973 1985

bcr

9

abl

22

Philadelphiachromosomeby t(9;22)

CBL

SOS

BCR-ABL

GRB-2P

Ras Raf-1

Mek1

ERK

P

P

MAPK

JUN

P

P

PI3K

AKT

P

P

Bcl-2

CRK

MYC

Survival Proliferation Migration

TalinF-aktin

FAK

Vinculin

P

P

P






BCR-ABLfusion gene

B. DrukerImatinib

1960 1973 1985 1995-2001

CBL

SOS

BCR-ABL

GRB-2P

Ras Raf-1

Mek1

ERK

P

P

MAPK

JUN

P

P

PI3K

AKT

P

P

Bcl-2

CRK

MYC

Survival Proliferation Migration

Talin

P

F-aktin

FAK

Vinculin

P

P

Imatinib


DeathBlast

crisisAccelerated

phase

Chronic

phase

3-5 years 3-6 months 3 months

Chronic

phase

Continuous treatment

Expensive

Pregnancy …


Blast crisis is rare, normal quality of life, long survival

Monitoring of the disease

Quantitative Real-Time PCR to measure BCR-ABL1 transcript levels

bcr

abl

BCR-ABL

Fusion gene Fusion mRNA Fusion protein

46%

0,07%

0,02%

0% 0% 0%0,001

0,01

0,1

1

10

100

1000

2007. április 2008. január 2008. május 2008. október 2009. március 2009. június

BC

R-A

BL%


Imatinib failure in ~25% of cases

88% 80% 41%

0,001

0,01

0,1

1

10

100

1000

2006.július 2007. március 2007. június

BC

R-A

BL

%

248%

0,5% 0,1%

0,05%

0,5%

6,5%

28% 42% 36%

0,001

0,01

0,1

1

10

100

1000

2007.jan.

2007.jún.

2007.aug.

2007.nov.

2008.febr.

2008.jún.

2008.aug.

2008.okt.

2009.febr.

2009.máj.

BC

R-A

BL%


Dasatinib NilotinibG250E F317L

Mutáció Dasatinib Nilotinib

Q252H

Y253H

E255K/V

V299L

T315I

T315A

F317L

F359V/C

Elimination of the dasatinib sensitive clone

Emergence of a novel mutation

Dynamics of resistance mutations

TARGETED THERAPY

How about the BCR-ABL negative MPNs ?

Chronic myeloid leukemia (CML)

Chronic neutropilic leukemia (CNL)

Polycythemia vera (PV)

Essential trombocythemia (ET)

Myelofibrosis (MF)

Chronic eosinophilic leukemia, NOS

Mastocytosis

Myeloproliferative neoplasms, unclassifiable

BCR-ABL1

c-KIT

Imatinib

Imatinib

The majority, if not all, of MPNs

are characterized by aberrant

tyrosine kinasesignalling

Genetic backgroundunknown until 2005

Myeloproliferative neoplasms

2005: Paris, Basel, Cambridge, Boston

James C, et al. Nature. 2005; 434:1144.

Kralovics R, et al. N Engl J Med. 2005; 352: 1779.

Baxter EJ, et al. Lancet. 2005; 365: 1054.

Levine R, et al, Cancer Cell 2005, 7: 387.

Activating JAK2 V617F mutation in PV, ET and MF

A breakthrough discovery, but the beginning of a journey ...

The JAK2 story …

P P

P

P

P

P

STAT5

MAP kinases

PI3K

Bcl-Xl

STAT5

STAT5

P

P

Anti-apoptotic signals

Survival

Differentiation

Proliferation

Quintas-Cardama, Nat Rev Drug Disc; 2011, doi:10.1038/nrd3264

The role of the JAK2 (Janus) kinase

The oncogenic JAK2 V617F mutation

N- -CKinasePseudokinaseSH2FERM

P P

James C, Nature 2005; 434: 1144-1148, A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera

V617F mutation

(1849G>T)

Constitutive activation

*

Mutation in the regulatory

pseudokinase domainPV (96%)

ET (~50%)

MF (~60%)

What is the true potential... ?

Ruxolitinib: approved

SAR302503: Phase III.

• Improvement in splenomegaly and systemic symptoms

• No improvement in BM fibrosis or reduction of mutant allele burden

• PV, ET patients: improvement in peripheral blood count

• Efficacy irrespective of the mutational status

• Side effects: anemia, thrombocytopenia, GI disturbances

• Long term side effects... ?

Issues in terms of selectivity, specificity

… not the magic bullets we were hoping for…

JAK2 inhibitor therapy ..

”bench to bedside: 2005-2012”

Genomic landscape of AML

Key cytogenetic events: t(15;17); t(8;21); inv(16) > favorable outcome

Key mutation events: FLT3-ITD, NPM1 and CEBPA

FLT3 ITD, in AML-NK NPM1 mutation in AML-NK

wt mut

FLT3-ITD mutation: poor prognosis NPM1 mutation: favorable prognosis

FLT3-ITD + / NPM1+ cases: poor prognosis

The changing genomic landscape of AML

Revolution in sequencing technologies (2006-…..)

100 machines, years, $$$$$$$$$

1 machine, weeks$$$

Next generationsequencing

The changing genomic landscape of AML

Mutation based outcome predictors

Patel et al, NEJM 2013; Grossmann et al, Blood 2013

Future genetic evaluation of AML …?

Godley et al, NEJM 2013

New promising (?) therapeutic targets….FLT3, DNMT3A, IDH1/2 …

Assessment of clonality in lymphoid malignancies

Immunoglobulin (IgH) gene rearrangement

Izotípusváltás (class switch)

5’

***

5’ 3’

V1 D 1-12V2 J1-4 CHVn

3’

germline Ig gene

V-D-J recombination

Every B-cell carries a unique Ig gene configuration

Huge variability >antigen recognition repertoire

Multiplex PCR

B-cell/IgH

Marker - NTC+Minta

Izotípusváltás (class switch)

5’

***

5’ 3’

V1 D 1-12V2 J1-4 CHVn

3’

germline Ig gene


Immunoglobulin (IgH) gene rearrangement

C.B.(19477, tube A)

C.B. (19557, tube A)

KA, 18129, TCR B

KA, 19178, TCR B


T-cell receptor (TCR) gene rearrangement analysis

Diffuse large B-cell lymphoma: molecular profiling (GC vs ABC)

Alizadeh et al, Nature 2000,

Patient Stratification:Remodel-B trial

B-cellreceptor

CD79 a/b activating mutations

SYKSYK

BTK

Toll-likereceptor

MYD88 activating mutations

PKC-b

CARD11

MY

D8

8

CARD11 activating mutations BCL6

MALT1

NFkBpathway

PI3K pathway

PI3K

MAP-K pathway

AKT

mTOR

Fostamatinib

Ibrutinib

CAL-101

Enzastaurin

mTOR i

MK2206

CD

79

aC

D7

9b

CD

79

bC

D7

9a

DLBCL: mutation profiles and novel targets

The Epigenetic Code

DNA methylation Histone modifications

• Methylation• Acetylation• Phosphorylation …

Attractive therapeutic targets … Proof of concept: DNA hypomethylating agents, HDACi

Specific enzymes Deposition of chromatin marks

Arrowsmith et al, Nature Reviews Drug Discovery, 11, 2012

DNMT

TET2 Alterations in cancer epigenome

HDACHAT

HMT KDM

Mutations in the epigenome of B-cell lymphomas

TRANSLATIONAL ASPECTS

Repression

Activation

EZH2MLL2

H3K27Me3

H3K4Me3

HDAC i

EZH2 i ?

H3K27Ac

P300

CREBBP

MUTATIONS IN THE EPIGENETIC MACHINERY

The changing (epi)genetic landscape of B-cell lymphomas- Novel mutations of epigenetic regulators (Pasqualucci, Nature, 2011)

H3K27me3 repressive mark

H3K4me3 activation mark

EZH2

MLL2

Gene Feature of mutation

Follicular lymphoma

Diffuse large B-cell lymphoma

Multiple myeloma

MLL2 loss of function 89% 32% 5%

EZH2 gain of function 15% 21% -

CREBBP loss of function 33% 25% -

P300 loss of function 15% 10% -

UTX loss of function - - 10%

FL: 90% !

Selective EZH2 inhibitors

Clinical Trial (2013) !

2010

2012 2012

NGS: Huge amount of novel data

Thank you for your attention !

internal medicine, yr 5 basic concept of the treatment of malignancies dank magdolna ... anemia,...

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