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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 bodor.csaba1@med.semmelweis-univ.hu
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
Chronic Myeloid Leukemia (CML)
Paradigm shift in oncology > targeted therapy
Philadelphia chromosome
t(9;22) translocation
BCR-ABLfusion gene
1960 1973 1985
Chronic Myeloid Leukemia (CML)
Paradigm shift in oncology > targeted therapy
Philadelphia chromosome
t(9;22) translocation
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
Chronic Myeloid Leukemia (CML)
Chronic Myeloid Leukemia (CML)
Paradigm shift in oncology > targeted therapy
Philadelphia chromosome
t(9;22) translocation
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
Chronic Myeloid Leukemia (CML)
DeathBlast
crisisAccelerated
phase
Chronic
phase
3-5 years 3-6 months 3 months
Chronic
phase
Continuous treatment
Expensive
Pregnancy …
Chronic Myeloid Leukemia (CML)
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%
Monitoring of the disease
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%
Monitoring of the disease
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”
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
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 …
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
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
Assessment of clonality in lymphoid malignancies
Immunoglobulin (IgH) gene rearrangement
C.B.(19477, tube A)
C.B. (19557, tube A)
KA, 18129, TCR B
KA, 19178, TCR B
Assessment of clonality in lymphoid malignancies
T-cell receptor (TCR) gene rearrangement analysis
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
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
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
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 !
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