4a. tirosin chinasi e mpd

WHO Integrated Classifications

Morphology:Cytology

Histopathology

Molecular GeneticsCytogenetics

Immunology:Immuno-cyto-histochemistry

HEMATOPOIESIS HEMATOPOIESIS

morfologiaPB

cariotipo

BCR/ABL1

ABL1/9q34BCR/22q11

Ph

t(9;22)(q34;q11)

BCR

ABL1 Ph-Ph+

NORMAL nuclei BCR/ABL1-positive nuclei

t(9;22) (q34;q11)

Indagini che identificano le cellulecon cromosoma Filadelfia

Metodica Potere di risoluzione

Citogenetica Convenzionale 1x102

FISH (Ibridazione in Situ con Fluorescenza) 1x103

RT-PCR 1x104 (105)

Diverse breakpoints/diverse fusion proteins

PCR analysis of BCR/ABL1 transcripts

- P190 transcript: Acute lymphoblastic leukemia (ALL)

- P210 transcript: chronic myeloid leukemia (CML)

- P230 transcript: chronic neutrophilic leukemia (CNL)

The ABL1 gene

•homologous to v-abl

•IN G1 it is bound by Rb1

•Knock-out mice are leukopenic

•when overexpressed --> cell cycle arrest

NLSACTIN

BINDINGSH1SH2SH3

DNA BINDING COOHNH2

ATPbind

NLS: Nuclear Localisation SignalTyr Kinase

ABL protein

Proteininteraction

The BCR gene

•Ubiquitous expression

•cytoplasmic, but pericromosomic during mitosis

•Knock-out have nearly normal hematopoiesis

PHSer/Trhkinase

DD DBL/GAP COOHNH2 177Y

Sh2 binding

BCR protein

Dimerization domain

Principali effetti di Bcr-Abl nel progenitore emopoietico

NUCLEO

Bcr-Abl 1. Stimolo alla proliferazione

4. Diminuzione della funzione di ABL a livello nucleare: instabilità genomica

Paxillin

F-actin

2. Diminuzione dell’adesione allo stroma emopoietico

Abl

3. Ridotta morte apoptotica

Ciclo cellulare

RAS

CITOPLASMA

NUCLEO

ABLTirosin Chinasi

TIROSIN CHINASI E NEOPLASIE MIELOIDI

Glivec (imatinib mesilato) Inibitore selettivo della tirosina-chinasi Bcr-Abl

La target therapy della LMC

Un approccio rivoluzionario

in terapia oncologica (target therapy): per la prima volta un farmaco che

riconosce ed elimina solo le cellule portatrici dell’anomalia

molecolare

TERAPIE MOLECOLARI(MIRATE/BERSAGLIO/TARGET/INTELLIGENTI)

BCR/ABL1 + loss 5'ABL1/3'BCR

normal BCR/ABL1

BCR/ABL1 + loss 5'ABL1

der(9)

Karyotype D-FISH

0

10

20

30

40

50

60

70

80

1 2 3 4 5 6 7

Serie1

0 1 2 4 8 12 17 months

FISH monitoring

IMATINIB: RESISTENZE

• Mutazioni nel dominio di tirosin-chinasi

• Amplificazione di BCR-ABL1

• Nuove abberrazioni cromosomiche

• alterato metabolismo dell’imatinib

Inibitori Tirosin chinasi

• Imatinib

• Nilotinib

• Dasatinib

• Sumitinib

• …………

• …………

Progressione della Leucemia Mieloide Cronica

N

NN

N

N

N

N

N

N

N

N

N

N

N

NN

N

N

N

N

N

N

N

NN

N

N

NN

NN

N N

N

NN

N

N

NN

N

N

N

N

NN

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NN

N

N

NN

N

Ph+

Ph+

Ph+

Ph+

Ph+

Ph+

Ph+ Ph+

Ph+Ph+

Ph+

Ph+

Ph+

Ph+Ph+

Ph+

Ph+Ph+

Ph+Ph+

Ph+

Ph+

Ph+

Ph+ Ph+

Ph+Ph+

Ph+

Ph+

Ph+ Ph+

Ph+Ph+

Ph+ Ph+ Ph+ Ph+ Ph+ Ph+

Ph+

Ph+Ph+

Ph+

Ph+

Ph+

Ph+

Ph+Ph+

Ph+

Ph+

Ph+

Ph+

Ph+

Ph+Ph+

Ph+

Ph+

Ph+Ph+

Ph+

Ph+

Ph+Ph+

Ph+Ph+

Ph+

Ph+

Ph+

Ph+

Ph+

Clone indifferenziato,crisi blastica

N = cellule normali

1 2 3 4 5

Adattata da Saglio G, Congresso SIE, Firenze 2001

Midollo osseo

Compartimento staminale

Anno

Chronic myelogenous leukemia

CMLBCR/ABL1

Others

PV

MFET

Myeloproliferative Disorders

HEMATOPOIESIS HEMATOPOIESIS

TYROSINE KINASE RECEPTOR FAMILY

Tyr Kinase domains

Cell Membrane

Extra Cellular

Intra Cellular

Phosphorilation - Dimerisation - Signal Transduction ( RAS; STAT; MAPK…)

RECEPTOR FAMILY

Tyr Kinase domains

Cell Membrane

Extra Cellular

Intra Cellular

Phosphorilation - Dimerisation - Signal Transduction ( RAS; STAT; MAPK…)

JAK 2MPL

ARG (ABL2) FLT3KIT

PDGFRαPDGFRβFGFR1

CITOPLASMA

NUCLEO

. ABL1

. JAK2 Tirosin-chinasi citoplasmatica

. PDGFRB

. PDGFRA

. FGFR1

. C-KITRecettori(Tirosin Chinasi)

TIROSIN-CHINASI E NEOPLASIE MIELOIDI CRONICHE

ST

AT

ST

AT

ST

AT

JAK

JAK-STAT PATHWAYJAK-STAT PATHWAY

NucleusNucleusCytosolCytosol STATSTAT

Plasma membranePlasma membrane

JAKJAK

ST

AT P P

P P

JAKJAKS

TA

T

ST

AT

ST

AT

ST

AT

ST

AT P

ST

AT

P

EPOEPO

EPOEPO

EPOEPO

EPOEPO

P

P

P

P

Ep

o-R

Ep

o-R

Ep

o-R

Ep

o-R

Ep

o-R

Ep

o-R

JAK

JAK-STAT PATHWAYJAK-STAT PATHWAY

NucleusNucleusCytosolCytosol STATSTAT

Plasma membranePlasma membrane

JAK-Val617FJAK-Val617F

ST

AT

ST

AT

ST

AT

ST

AT

ST

AT P

ST

AT

P

P

P

P

Ep

o-R

Ep

o-R

ST

AT

ST

AT

ST

AT

ST

AT P P

P P

Ep

o-R

Ep

o-R

JAK-Val617FJAK-Val617F

JAK-Val617FJAK-Val617F JAK-Val617FJAK-Val617F

P

JAK2 V617F

MUTAZIONI DI JAK2(Activating point V617F mutation)

Esone 14 JAK2:

G sostituita da una T (nucleotide 1849)

Valina sostituita da Fenilalanina (aminoacido 617)

Mutazione somatica: cellula staminale pluripotente eterozigote o omozigote attivazione costitutiva di

JAK2

MPL somatic mutation in CMPD

W: triptofanoL: leucinaK: lisina

C-MPL mutations: TGG TTG conversion in MPLW515L

TGG AAG conversion in MPLW515K

transmembrane domain mut

C-MPL mutations may occur with JAK2V617F

Frequency: 5-10%

Diseases: PMF and ET

CMPD

W: triptofanoL: leucinaK: lisina

CMLBCR/ABL1

Others

PV

MFET


JAK2 MPL

CITOPLASMA

NUCLEO

Recettore(Tirosin Chinasi)


CMLBCR/ABL1

Others

PV

MFET


JAK2 MPL

Proliferation

RAS

GRB-2

SOS

PDGFRLigand PDGFR HLH

PDGFRα 4q12PDGFRβ 5q33

Tyrosin kinase domain

MPD Ph negative (eosinophilia; monocytosis)

t(5;12)(q33;p13)

PDGFR gene

HLH DNA-bindingN C

ETV6 gene

t(5;12)(q33;p13)-ETV6/PDGFR

Diagnosi: Ph- CML CMML MDS

Eosinofilia BM/PB

Monocitosi PB>8%

Risposta al trattamento con Imatinib Mesilato

7q11/HIP1

14q22/NIN

14q32/KIAA1509

1p36?

17p11/HCMOGT-1

12p13/ETV6

17p13/RABEP1

14q32/CEV14

10q22/H4

16p13/?15q22/TP53BP1(RABPT5)

12q13/?

2q37/?

3p21/?

1q23/PDE4DIP

1q21/?????

PDGFRB

Geni partners di PDGFRB nei disordini Mieloproliferativi cronici Ph-

NH2 COOH

PDGFRBGene Partner

UN GENERICOMBINAZIONI

GENOMICHEMULTIPLE

UNA MALATTIA

Proliferation

RAS

GRB-2

SOS

PDGFRLigand PDGFR HLH

PDGFRα 4q12PDGFRβ 5q33

Tyrosin kinase domain

Reattiva non clonale

EOSINOFILIA

Sindrome ipereosinofila idiopatica / leucemia eosinofila cronica: HES/CEL

REATTIVA NON CLONALE

INFEZIONI E PARASSITOSI

MALATTIE ALLERGICHE

MALATTIE DEL CONNETTIVO E AUTOIMMUNI

PROCESSI NEOPLASTICI

1. conta degli eosinofili nel sangue persistentemente

superiore a 1,5 x109/l per un periodo superiore a sei

mesi

2. segni e/o sintomi di coinvolgimento di organi

3. assenza di una causa nota o di un’anomalia clonale

HES

I 3 criteri di definizione della HES in base alla WHO sono:

RP11-3H20

FIP1L1 CHIC2 PDGFRA

telcen

telcenFIP1L1/PDGFRA

Hypereosinophilic Syndrome, Myeloproliferative Variant

4q12

I-FISH 34 pts with HES

FIP1L1/PDGFR+ FIP1L1/PDGFR-

PTS 8 26

M/F 7/1 12/14

Median age 42 51

Hepatomegaly 6 5

Splenomagaly 6 6

Hearth 2 3

CNS 2 3

Lung 1 3

Skin 4 10

Response toSTI571

7/7 0/4

WHO Classification of Systemic Mastocytosis

Mast cell leukemia

Aggressive systemic mastocytosis

SM with an associated Haematopoietic clonal non-mast cell lineage disease

Indolent Systemic Mastocytosis ( Isolated bone marrow mastocytosis;

Smouldering systemic mastocytosis )

Variants and subvariants

FIP1L1 PDGFRACHIC2(Deletion)

KIT(Mutations)

5’ 3’ 3’5’centromero telomero

C-Kit activation loop: D816V (aspartic acid to valine at aa 816)

D816Y (aspartic acid to tyrosine at aa 816)

Resistence: imatinib mesilate (in vitro and in vivo)

Sensitivity: PK412 (in vitro)

C-KIT mutations

Systemic mast cell disease / acute myeloid leukemia

CITOPLASMA

NUCLEO


PDGFRBPDGFRAFGFR1KIT


47,XX,t(8;13)(p11.2;q12q13),+21

der(13)

der(8)813

t(8;13)(p11;q12)-ZNF198/FGFR1

8p11

centromere telomereFGFR1

RP11-350N15

5’ 3’

t(6;8)(q27;p11)/FOP-FGFR1: MPD/NHL(Popovici C et al., Blood 1999)

t(8;9)(p11;q33)/CEP110/FGFR1: MPD/NHL (Guash G et al, Blood 2000)

ins(12;8)(p11;p11p22)/FGFR1OP2-FGFR1: MPD/NHL (Grand EK et al., GCC 2004)

t(8;13)(p11;q12)/ZNF198-FGFR1: MPD/NHL (Reiter A et al., Blood 1998)

t(8;19)(p11;q13)/HERVK-FGFR1: MPD/NHL (Guash G et al., Blood 2003)

t(8;22)(p11;q11)/BCR-FGFR1: CML (Demiroglu A et al., Blood 2001)

t(7;8)(q32;p11)/TRIM24-FGFR1 (Belloni E et al., Genes Chrom Cancer 2005)

t(8;17)(p11;q11)/MYO18A-FGFR1 (Belloni E et al., Genes Chrom Cancer 2005)

t(8;9)(p22;p24)/PCM1-JAK2 (Adelaide J et al., Leukemia2006)

Rearrangements with eosinophilia

fusion partners

FGFR1 13q12/ZNF198

9q34/CEP110 12p11/FGFG1OP2

22q11/BCR

Ig1 Ig1 transmembraneN CIg1 Ig2 Ig3 TM TK1 TK2

Ig-like domainstransmembrane

tyrosin kinase

19q13/HERV-K

6q26/FOP

CITOPLASMA

NUCLEO

ABL ; JAK2 Tirosin Chinasi


PDGFRBPDGFRAFGFR1KIT


1. Chronic myeloproliferative diseases

- Chronic myelogenous leukemia

- Chronic neutrophilic leukemia

- CEL/ hypereosinophilic syndromePDGFRA

- Polycythemia vera JAK2

- Chronic idiopathic myelofibrosis JAK2

- Essential thrombocythemia JAK2

-Chronic myeloproliferative disease, unclassifiable PDGFRB

-Systemic Mastocytosis KIT

Ph+

Ph-

L’IMATINIB MESILATO PUO’ ESSERE UNA VALIDA

TERAPIA NEL TRATTAMENTO DEL DANNO D’ORGANO

In 1 paziente con lesioni del SNC dopo undici mesi di terapia

si verificava una riduzione delle lesioni encefaliche

Campbell P and Green A. N Engl J Med 2006;355:2452-2466

Role of JAK2 in Pathway Signaling and Erythropoietin Binding, Stem-Cell Differentiation, and Development of Homozygosity for the V617F

Mutation

EPO-dependent signal EPO-independent signal

EPO, TPO, G-CSF, GM-CSF, IL3, IL5: cytokines using JAK2 for signaling transduction

Acquired PDGFRA mutations with resistance to imatinib

Kinase domain (ATP- binding region)

T674I (treonine to isoleucine at aa 674)

Resistence to imatinib therapy

RP11-110K18(5’ZNF198) +RP11-350N15 (FGFR1)

The 8p12 myeloproliferative syndrome8p12 MPS

Myeloid hyperplasia

Eosinophilia /Monocytosis

Associated T (B)-cell lymphoma

Progression toward AML

centromere telomereCSF1R PDGFRB CDX1

5q33

cosmid 9-4 cosmid 4-1

5’3’

t(1;5)(q23;q33)/PDE4DIP- PDGFR: MDS/MPD (Wilkinson K et al., Blood 2003)

t(5;7)(q33;q11)/HIP1-PDGFR: CMML (Ross TS et al., Blood 1998)

t(5;10)(q33;q22)/ H4-PDGFR: aCML (Schwaller J et al., Blood 2002)

t(5;12)(q33;p13)/ETV6/-PDGFR: aCML, CMML, RAEB (Golub R et al., Cell 1994)

t(5;14)(q33;q24)/NIN- PDGFR: MPD (Vizmanos JL, et al., Cancer Res 2004)

t(5;14)(q33;q32)/CEV14- PDGFR: AML (Abe A et al., Blood 1997)

t(5;17)(q33;p13)/RABAPTIN-5- PDGFR: CMML (Magnusson MK et al., Blood 2001)

Rearrangements with eosinophilia

Loss of heterozygosity of chromosome 9 – occurs commonly in P vera

normal LOH

p24 JAK2

Characteristics of bcr-abl

•Only cytoplasmic

•permanently activated

•several targets

•it mimics some TK receptors, inducing constitutive activation of several pathways

•bypass of regulatory mechanisms

4a. tirosin chinasi e mpd

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