WHO Integrated Classifications

Morphology: Immunology:
Cytology Immuno-cyto-
Histopathology histochemistry

Molecular
Cytogenetics Genetics
HEMATOPOIESIS
HEMATOPOIESIS
morfologia
PB
cariotipo
 BCR/22q11 ABL1/9q34

Ph
BCR/ABL1

t(9;22)(q34;q11)
 t(9;22) (q34;q11)

NORMAL nuclei BCR/ABL1-positive nuclei

BCR

ABL1 Ph-
Ph+
 Indagini che identificano le cellule
con 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

ABL protein
ATP
bind

NH2 DNA ACTIN

SH3 SH2 SH1 NLS COOH
BINDING BINDING

Protein
Tyr Kinase NLS: Nuclear Localisation Signal
interaction
 The BCR gene

•Ubiquitous expression
•cytoplasmic, but pericromosomic during mitosis
•Knock-out have nearly normal hematopoiesis

BCR protein

NH2 Ser/Trh
DD 177
Y DBL/GAP PH COOH
kinase

Dimerization Sh2 binding

domain
Principali effetti di Bcr-Abl nel progenitore emopoietico

RAS
Bcr-Abl 1. Stimolo alla proliferazione

F-actin NUCLEO

Paxillin Ciclo
Abl cellulare

2. Diminuzione 4. Diminuzione della funzione

dell’adesione allo 3. Ridotta morte di ABL a livello nucleare:
stroma emopoietico apoptotica instabilità genomica
TIROSIN CHINASI E NEOPLASIE MIELOIDI

CITOPLASMA

ABL
Tirosin Chinasi

NUCLEO
 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)
 TERAPIE MOLECOLARI
(MIRATE/BERSAGLIO/TARGET/INTELLIGENTI)
 Karyotype D-FISH
normal BCR/ABL1

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

der(9)

80
70
60
50
FISH monitoring 40 Serie1

30
20
10
0
01 2
1 23 44 58 6
12 7
17 months
 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
Anno

1 2 3 4 5

N N N N
N N N Ph+
N N N N N
N N N
N N Ph+ Ph+
N N N N
N N N N Ph+
N N N N Ph+ Ph+ Ph+
N
N N N N
N N N Ph+ Ph+
N N
N N NN Ph+ Ph+
N
N N Ph+ Ph+
N N N Ph+
N N N Ph+ Ph+
N N N Ph+ Ph+
N N N N Ph+ Clone
Ph+ indifferenziato,
Ph+
N N Ph+ Ph+
N
Ph+ crisi blastica Ph+
N N N Ph+
N Ph+ Ph+ Ph+
N Ph+ Ph+
N Ph+ Ph+ Ph+ Ph+ Ph+
N
N Ph+ Ph+ Ph+
Ph+ Ph+ Ph+
N Ph+ Ph+ Ph+
Ph+ Ph+ Ph+
Ph+ Ph+ Ph+ Ph+ Midollo osseo
Ph+ Ph+ Ph+
Ph+ Ph+ Ph+ Ph+ Ph+ Ph+
Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Ph+ Compartimento
Ph+ staminale
N = cellule normali

Adattata da Saglio G, Congresso SIE, Firenze 2001

Chronic myelogenous leukemia
Myeloproliferative Disorders

ET
MF

PV

Others
CML
BCR/ABL1
HEMATOPOIESIS
HEMATOPOIESIS
TYROSINE KINASE
RECEPTOR FAMILY
Extra Cellular

Cell Membrane

Tyr Kinase Intra Cellular

domains

Phosphorilation - Dimerisation -
Signal Transduction ( RAS; STAT; MAPK…)
 JAK 2
MPL
RECEPTOR FAMILY ARG (ABL2)
FLT3
Extra Cellular KIT
PDGFRα
PDGFRβ
FGFR1

Cell Membrane

Tyr Kinase Intra Cellular

domains

Phosphorilation - Dimerisation -
Signal Transduction ( RAS; STAT; MAPK…)
TIROSIN-CHINASI E NEOPLASIE MIELOIDI CRONICHE

CITOPLASMA

. PDGFRB
. ABL1 . PDGFRA
. FGFR1
. JAK2 . C-KIT
Tirosin-chinasi citoplasmatica Recettori
(Tirosin Chinasi)

NUCLEO
 JAK-STAT PATHWAY
EPO EPO

EPO EPO

Epo-R
Epo-R
Epo-R
Epo-R
Epo-R

Epo-R

Plasma membrane

STAT

STAT
P P

P P JAK JAK

STAT
STAT
P P
JAK JAK STAT

P P
JA
K
JA

STAT

STAT
P
STAT

STAT

STAT
P
STAT
Cytosol Nucleus
 JAK-STAT PATHWAY

Epo-R
Epo-R
Epo-R
Epo-R Plasma membrane

STAT

STAT
P P

P P JAK-Val617F JAK-Val617F

STAT

STAT
JAK-Val617F JAK-Val617F P P

P P
STAT
STAT

STAT
P
STAT

STAT

STAT
P
STAT
Cytosol Nucleus
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: triptofano
L: leucina
K: lisina
 CMPD

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

W: triptofano
L: leucina
K: lisina
 Myeloproliferative Disorders
MPL
JAK2
ET
MF

PV

Others
CML
BCR/ABL1
TIROSIN CHINASI E NEOPLASIE MIELOIDI

CITOPLASMA

Recettore
(Tirosin Chinasi)

NUCLEO
 Myeloproliferative Disorders
MPL
JAK2
ET
MF

PV

Others
CML
BCR/ABL1
 Ligand PDGFR HLH
PDGFR

Tyrosin kinase domain

GRB-2

SOS

RAS
PDGFRα 4q12
PDGFRβ 5q33 Proliferation
t(5;12)(q33;p13)
MPD Ph negative
(eosinophilia; monocytosis)

N HLH DNA-binding C

ETV6 gene

PDGFR 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

Geni partners di PDGFRB nei disordini Mieloproliferativi
cronici Ph-

7q11/HIP1
3p21/? 10q22/H4
2q37/? 12p13/ETV6

1p36? 12q13/?
PDGFRB 14q22/NIN
1q23/PDE4DIP
14q32/CEV14
1q21/?????
14q32/KIAA1509
17p11/HCMOGT-1 15q22/TP53BP1(RABPT5)
17p13/RABEP1 16p13/?

NH2 COOH

Gene Partner PDGFRB

 RICOMBINAZIONI
UN GENE GENOMICHE UNA MALATTIA
MULTIPLE
 Ligand PDGFR HLH
PDGFR

Tyrosin kinase domain

GRB-2

SOS

RAS
PDGFRα 4q12
PDGFRβ 5q33 Proliferation
 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
 HES

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

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

Hypereosinophilic Syndrome, Myeloproliferative Variant

FIP1L1 CHIC2 PDGFRA

4q12 cen tel
RP11-3H20

cen tel
FIP1L1/PDGFRA
 I-FISH 34 pts with HES

FIP1L1/PDGFR+ FIP1L1/PDGFR-

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 to 7/7 0/4
STI571
 WHO Classification of Systemic Mastocytosis

Variants and subvariants

Indolent Systemic Mastocytosis

( Isolated bone marrow mastocytosis;

Smouldering systemic mastocytosis )
SM with an associated Haematopoietic
clonal non-mast cell lineage disease

Aggressive systemic mastocytosis

Mast cell leukemia

5’ 3’ 5’ 3’
centromero telomero
FIP1L1 CHIC2 PDGFRA KIT
(Deletion (Mutations)
 C-KIT mutations

Systemic mast cell disease / acute myeloid leukemia

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)

TIROSIN CHINASI E NEOPLASIE MIELOIDI

CITOPLASMA

Recettore
(Tirosin Chinasi)

PDGFRB
PDGFRA
NUCLEO FGFR1
KIT
47,XX,t(8;13)(p11.2;q12q13),+21
t(8;13)(p11;q12)-ZNF198/FGFR1

der(13)
13
8 der(8)
 8p11
RP11-350N15

FGFR1
centromere 5’ 3’
telomere

Rearrangements with eosinophilia

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)
 fusion partners

9q34/CEP110 12p11/FGFG1OP2

6q26/FOP 13q12/ZNF198
FGFR1

22q11/BCR 19q13/HERV-K

Ig-like domains tyrosin kinase

transmembrane

N Ig1 Ig2 Ig1 Ig1TM

Ig3 transmembrane
TK1 TK2 C
TIROSIN CHINASI E NEOPLASIE MIELOIDI

CITOPLASMA

Recettore
ABL ; JAK2 (Tirosin Chinasi)
Tirosin Chinasi

PDGFRB
PDGFRA
NUCLEO FGFR1
KIT
1. Chronic myeloproliferative diseases

- Chronic myelogenous leukemia

Ph+
- Chronic neutrophilic leukemia
- CEL/ hypereosinophilic syndromePDGFRA
- Polycythemia vera JAK2
- Chronic idiopathic myelofibrosis JAK2 Ph-
- Essential thrombocythemia JAK2
-Chronic myeloproliferative disease, unclassifiable PDGFRB
-Systemic Mastocytosis KIT
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
 Role of JAK2 in Pathway Signaling and Erythropoietin Binding, Stem-
Cell Differentiation, and Development of Homozygosity for the V617F
Mutation

Campbell P and Green A. N

Engl J Med 2006;355:2452-2466

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 syndrome
8p12 MPS

Myeloid hyperplasia
Eosinophilia /Monocytosis
Associated T (B)-cell lymphoma
Progression toward AML
 5q33

cosmid 9-4 cosmid 4-1

CSF1R PDGFRB CDX1

centromere 3’ 5’ telomere

Rearrangements with eosinophilia

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

p24 JAK2

normal LOH
 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