TUMORI SOLIDI

CARCINOMA DEL POLMONE

 Angiogenesis

growth of new vessels from existing vasculature

Physiological – wound healing,

embryogenesis, menstruation

Pathological – tumour growth,

rheumatoid arthritis, psoriasis
 Adhesion to vessel
wall in distant organ
invasion angiogenesis intravasation

Extravasation and migration micrometastasis metastasis

 Steps in Angiogenesis

1. the release of proteases from "activated" endothelial

cells
2. degradation of the basement membrane surrounding
the existing vessel
3. migration of the endothelial cells into the interstitial
space
4. endothelial cell proliferation
5. lumen formation
6. generation of new basement membrane with the
recruitment of pericytes
7. fusion of the newly formed vessels
8. initiation of blood flow.
The angiogenic switch hypothesis - a balance
between pro- and anti-angiogenic factors

Hanahan and Folkman, 1996. Cell 86:353

The tumour and its surrounding environment are
known to promote VEGF release

Hypoxia PDGF
IGF-1
EGF

IL-8
Binding and activation
VEGF release of VEGF receptor-2
bFGF

COX-2
Nitric oxide
Oncogenes

P– –P
P– –P

Survival Proliferation Migration

Permeability
ANGIOGENESIS
IGF = insulin-like growth factor; PDGF = platelet-derived growth factor;
EGF = epidermal growth factor
The VEGF family and its receptors

VEGF-A
VEGF-B VEGF-A
PlGF VEGF-C
VEGF-D
VEGF
VEGF receptor-2
receptor-1 VEGF
receptor-3
P– –P
–P P– –P P–
P– –P P– –P
P– –P

Migration, permeability, DNA synthesis, survival

Angiogenesis Lymphangiogenesis
 VEGF Receptor Activation

Receptor
Receptor dimerization
dimerization
Autophosphorylation
Autophosphorylation

Dimeric
Dimeric
VEGF
VEGF ligand
ligand
Adapter
Adapter molecule
molecule binding
binding
p85
p85

PLC
PLC GRB2
GRB2

Ligand
Ligand Permeability
Permeability
binding
binding Downstream
Downstream
Proliferation
Proliferation
Migration signaling
signaling events
events
Migration
Adhesion
Adhesion Nucleus
Nucleus
Survival
Survival
Transcription
Transcription
Endothelial
Endothelial cell
cell
Strategies to inhibit VEGF signalling

Ferrara & Kerbel Nature 438: 967–974, 2005.

 Bevacizumab (Avastin)

VEGF isoforms recognised by

hypervariable murine
antibody fragment

Human
IgG-1
L’anticorpo anti-VEGF bevacizumab blocca
l’interazione del VEGF con i suoi recettori
VEGF
 Bevacizumab si lega al
Bevacizumab
VEGF bloccando
l’interazione con i
recettori e l’attivazione X
della trasduzione del
segnale a valle P– –P
P– –P

 Il blocco del VEGF

X
induce la regressione
Crescita
della vascolarizzazione
Proliferazione
tumorale
Migrazione
Sopravvivenza
 Attività biologiche del VEGF

 
mitogeno in vitro per diversi tipi di cellule endoteliali
induttore di angiogenesi in vivo
fattore di sopravvivenza cellulare in vitro
fattore di permeabilità vascolare
 stimola la migrazione cellulare
 inibisce l’apoptosi
 ERB
The ErbB Family receptors

4 types of erbB receptor 11 Ligands each with a common

EGF like structure
ErbB1 (HER1, EGFR) EGF

ErbB2 (HER2, neu) TGF-a

Amphiregulin
ErbB3 (HER3) Betacellulin
ErbB4 (HER4) EGF Epiregulin
HB-EGF
Epigen
Neuregulin 1 (NRG1)
Neuregulin 2 (NRG2)
Neuregulin 3 (NRG3)
Neuregulin 4 (NRG4)
 Espressione di EGFR nei tessuti normali

Cute, apparato digerente, sistema respiratorio,

ghiandole mammarie, apparato genitale, apparato
urinario, SNC, sistema endocrino

E’ presente sulle:
– cellule epiteliali
– cellule stromali
– cellule gliali
– cellule muscolari lisce
EGFR: FUNZIONE FISIOLOGICA

Una delle principali vie

molecolari attivate dall’EGFR è
quella di ras. Ras viene reclutata
attraverso il legame con la
molecola SOS.
EGFR: FUNZIONE FISIOLOGICA

A sua volta, ras determina

l’attivazione della
serina/treonina chinasi raf,
delle MAPKK 1 e 2 e delle
MAPK ERK 1 e 2.
EGFR: FUNZIONE FISIOLOGICA

Il risultato dell’attivazione di
questa via molecolare è
l’espressione di varie proteine
nucleari, inclusa la ciclina D1
(necessaria per il passaggio
G1-S nel ciclo cellulare).
 Strategie anti-EGFR
Anticorpi monoclonali

TK TK Piccole molecole anti-TK

PI3-K pY pY GRB2
pY SOS
STAT3 RAS RAF
PTEN AKT
MEK

Gene transcription
MAPK
G1
M S
Proliferation/ Survival
maturation
G2 (anti-apoptosis)
Chemotherapy /
radiotherapy Metastasis
Angiogenesis
resistance
 Ligand Extracellular
Ligand binding
Domain
Membrane

Tyrosine Kinase
PI 3 Kinase
Domain
P85
P P Intracellular
SHC P100

GROWTH, DIFFERENTIATION, APOPTOSIS ETC

Summary of EGFR mutations (PNAS 2004 101 13306)
 A review of 3016 cases from the literature.
Chan, Hill and Gullick EJC 2006 42 17.

688 728 729 761 762 823 824 875

250
No. of times observed

200

150

100

50

0
)
9 A 9K 9A 9S OP 50) 51) (V) (D ) (P) 51) (S) H 51) sN 4R (Y) 3L Q) V) (G) H ) 5Y 0M 9F 5L 6R 8R 1G
0 0 71 71 T -7 -7 ns ns ns -7 ns s(Q -7 )in 5 70 77 AF AS 1 NP 77 9 77 83 84 85 87
E7 E7 G G 31S 46 46 1) i 2) i 9) i 47 1) i ) in 49 59 K7 s7 H (E 70( s77 74( H T7 G H K L A
7 l(7 l(7 -75 -75 -74 l(7 -75 52 l(7 1-7 ; in 6 1 7
s i n 7
s
W de d e 6
4 4 6 47 de 47 -7 de (75 0G n s7 i n in
7 l 7 i
l(7 l(7 l(7 l(7 74 de 7
de de de d e de l ( D
 Mutations are associated with “Never-
Smokers”, gender and Histological subtypes

• Mutations are more common in East Asians (30.6% than in

Caucasians (7.6%) p<0.0001
• Mutations are more common in non-smokers (34.8%) than
in smokers (7.8%) p<0.0001
• Mutations are more common in women (26.4%) than in men
(9.3%) p<0.0001
• Mutations are more common in adenocarcinomas (23.2%)
or bronchioalveolar carcinoma (17.9%) than in other
histologies (2.2%)
Incidence of Kinase Mutations in Other Tumour Types
Hepatocellular 0/73 Lee et al. 2005

Pancreatic 2/55 (4%) Kwak et al. 2006

Esophageal 2/17 (12%) Kwak et al. 2006

Barret’s 3/21 (14%) Kwak et al. 2006
Colon 0/98 Lee et al. 2005

Breast 0/42 Generali et al. 2007

0/15 Lynch et al. 2004
0/11 Bargava ert al. 2005
1/93 Lee et al. 2005
Head & Neck 17/108 (16%) Na et al. 2007
1/100 (1%) Leoffler-Ragg et al.
2005
3/41 (7%) Lee et al. 2005
Cholangeocarcinoma 3/22 (14%) Gwak et al. 2005

Acute Adult Leukaemia 0/88 Lee et al. 2005

Gastric 0/185 Lee et al. 2005

Other 9/566 (2%) Sihto et al. 2005

 Current receptor inhibitors

• Monoclonal
antibodies

• Small molecule
RTK inhibitors
 HER family: targeted approaches

R R
mAb External domain

K K Small mol. Internal domain

Tumor response
Lung
Ovary
Prostate
Gastric
Breast Colon
 Responses in patients with or
without mutations

Patients with NSCLC responsive to gefitinib or

erlotininb are more likely to harbour mutations than
not (76.7% vs 23.3%)

However 12.3% of patients with mutations progress on

TKIs and 86.8% achieving stable disease possess wt
EGFR
Association of Site of Mutation and Sensitivity
to TKI treatment

85-90%
Total

Riely et al. Clin Cancer Res 2006 12 7232

Development of Drug Resistance
Secondary mutations in the EGFR
conferring drug resistance
 Kobayashi et al. NEJM 24/2/05

• A non-small-cell lung cancer that

was highly responsive to
gefitinib contained a mutation in
the epidermal growth factor
receptor (EGFR) gene that
increases susceptibility of the
tumor to gefitinib

• After two years of remission, the

disease relapsed

• A second biopsy of the tumor

revealed a new mutation in the
gene that negated the effects of
gefitinib
 EML4-ALK fusion in NSCLC

• Detected in a 62 years old Japanese male

with resected adenocarcinoma

• Tobacco smoker

• Wild type Kras and EGFR

Soda et al. Nature (2007) 448: 561-6

NSCLC: NON SMALL CELL LUNG CARCINOMA
 EML4-ALK fusion in NSCLC

• N terminal portion of EML4 was

fused to the intracellular kinase
domain of ALK

• Both map to the short arm of

chromosome 2 (2p21 and 2p23
respectively)

Soda et al. Nature (2007) 448: 561-6

 EML4-ALK variants
• Multiple variants of EML4-ALK
noted depending on the break
point on the EML gene

• Variable truncations of EML4

(occurring at exons 2, 6, 13, 14,
15, 18, and 20)

• ALK fusion starts at a portion

encoded by exon 20

Horn et al. J Clin Oncol 27(26):4232-5

 Frequency of EML4-ALK

• 5 out of 75 NSCLC tumor

samples were EML4-ALK (+)

• Not detected in 261 non lung

tumor specimens

Soda et al. Nature (2007) 448: 561-6

 ALK
• ALK is a tyrosin-kinase receptor

• ALK is expressed in brain and testis

• ALK was identified as a fusion partner of NPM1 in

anaplastic large-cell lymphoma with t(2;5)

• Since then fusion genes involving ALK have been

described in ALCL, inflammatory myofibroblastic
tumors and neuroblastomas
 EML4-ALK

• Wild-type ALK is thought

to be activated in response
to binding of a specific
ligand

• EML4–ALK is oligomerized
via the coiled coil domain
(CC) of EML4

• Resulting in persistent
mitogenic signaling that
leads to malignant
transformation.
 Transforming ability of EML4-ALK

• Expression plasmids for

EML4–ALK

• Introduced it into mouse

3T3 fibroblasts

• Subcutaneous injection
of the transfected 3T3
cells into nude mice
formed tumors

Soda et al. Nature (2007) 448: 561-6

Soda et al. PNAS (2008) 106:19893-7
 Conclusion

• EML4-ALK fusion is rare event in NSCLC (5-10%)

• Present in both Asians and North
American/European population
• Exclusive to NSCLC
• More common in never smokers
• ALK inhibitors may have a role in the treatment of
NSCLC with EML4-ALK fusion
CARCINOMA DELLA MAMMELLA
 Role of HER2 in breast cancer

 HER2 gene amplification or receptor overexpression

occurs in approximately 30% of metastatic breast
cancers

 HER2-positive tumours are associated with poor

prognosis and shortened disease-free/overall
survival

 HER2 receptor provides an extracellular target

for novel and specific anticancer treatment
 Transmembrane structure of
HER2 monomer

Extracellular domain
(632 amino acids)
Ligand-binding site

Transmembrane domain
(22 amino acids)
Plasma
membrane
Intracellular domain
(580 amino acids)
Tyrosine kinase activity
Cytoplasm
 HER2 receptor dimer transmembrane signal
transduction pathway

Growth factor

Binding site

Plasma
membrane
Tyrosine
Signal kinase activity
transduction
to nucleus

Cytoplasm Nucleus

CELL
Gene activation
DIVISION
 Indicators of increased HER2 production

Normal Amplification/overexpression

Cytoplasmic
3
membrane
C

B 2
1
A Nucleus 4

Cytoplasm

1 = ­ gene copy number

A = HER2 DNA 2 = ­ mRNA transcription
B = HER2 RNA 3 = ­ cell surface receptor protein expression
C = HER2 receptor protein 4 = ­ release of receptor extracellular domain
 HER2 technical approaches

 Gene amplification

– FISH/CISH

 Protein over-expression
– immunohistochemistry
0 1+ 2+ 3+

HER2-positive IHC stain

Dual-color FISH
 HER2 amplification by FISH

No amplification Amplification
 Her2 testing algorithm
Patient tumour
sample
IHC FISH

2+ 3+ + –

Retest with Trastuzumab Trastuzumab

FISH therapy therapy

– +

Trastuzumab
therapy
 Trastuzumab:
humanised anti-HER2 monoclonal antibody
• Bersaglio:
l’oncoproteina HER2
• Alta affinita’ (Kd=0.1nM)
e specificita’
• 95% umano, 5% murino
– Diminuito potenziale
immunogeno
– Aumentato potenziale per
arruolare meccanismi
effettori immuni
Trastuzumab e altri anticorpi monoclonali impediscono la
etero-dimerizzazione EGFR-HER2 e HER2-HER3

HER2
Ligand-binding domain
(inactive)

Pertuzumab

Trastuzumab

Cell membrane

Tyrosine kinase domain

 Piccole molecole ad attività anti-
tirosinchinasi che blocca HER1 ed HER2
 Inibitori tirosin-chinasici
F

Cl F Cl O

N N N
N N N N N N
H H H

O O O O O

O O HN
N

O
S
O O

Erlotinib Gefitinib Lapatinib

Small head group quinazolines Large head group quinazoline

 Breast Cancer

• 70-80% sporadici
• 15-20% familiari
• 5-10% ereditari
 TUMORE DELLA MAMMELLA FAMILIARE

Condizione di predisposizione ereditaria:

• più casi di carcinoma alla mammella in parenti di primo grado

soprattutto in giovane età o con un coinvolgimento di
entrambe le mammelle

• carcinoma dell’ovaio in familiari di primo grado

• casi di carcinoma della mammella maschile (BRCA2)

TUMORE DELLA MAMMELLA FAMILIARE

Nel 1994-1995 sono stati identificati due

geni responsabili della suscettibilità al
cancro della mammella: BRCA1, sul
cromosoma 17, e BRCA2 sul cromosoma
13.

Gli individui portatori di mutazioni a carico

di uno dei due geni hanno un rischio
maggiore di insorgenza di tumore alla
mammella o tumore ovarico ad un certo
punto della loro vita.
 BRCA1/BRCA2

ONCOSOPPRESSORI

BRCA1 (17q21) e BRCA2 (13q12) sono collegate ed integrate in alcuni

fondamentali “pathway” di “risposta al danno del DNA”.

FUNZIONI

• inibiscono la crescita delle ghiandole mammarie

• interagiscono con le proteine RAD (proteine del riparo)

• regolano l’attività di altri geni (p21) (proteina regolatrice della

trascrizione del DNA

• giocano un ruolo fondamentale nell’embriogenesi

 BRCA1/BRCA2

Sono state identificate oltre 600 mutazioni (proteine

tronche, inattivazione genica) in BRCA1 e BRCA2

ANALISI MUTAZIONALE DI TUTTA LA SEQUENZA GENICA

BRCA1: 24 esoni (ATG in ex2) Test diagnostico:

BRCA2: 27 esoni (ATG in ex2) Campione di PB
BRCA1/17q21

BRCA2/13q12
Pedigree of a family with germline BRCA2: 6503delTT mutation

father mother

proband

BRCA2 mut
CARCINOMA DEL COLON EREDITARIO

HNPCC: hereditary non-polyposis colorectal cancer

FAP: Familial Adenomatous Polyposis

 HNPCC: CARCINOMA DEL COLON.-RETTO FAMILIARE NON
POLIPOSICO

Clinical Presentation

• Earlier than average age of onset – mean 45

• Pattern of primary cancers segregating in family
• Distinguishing pathological features
• CRC usually involves the proximal colon
• Increased incidence of synchronous and metachronous CRC
• Survival rate for CRC appears better than that of it’s sporadic
variant
• Extra colonic cancers (Lynch II)
 HNPCC: CARCINOMA DEL COLON-RETTO FAMILIARE NON
POLIPOSICO

Pathology

• Poorly differentiated, mucinous and increased incidence of signet

cells
• Diploid, peritumoral lymphocyte infiltration and Crohn’s like reaction
• Adenomas found in 20% of colons in HNPCC patients with CRC
• Colonic adenomas tend to occur earlier, are larger and more often
villous with more high grade dysplasia
• Accelerated rate of adenoma to carcinoma
 FAP: Familial Adenomatous Polyposis

1. Epitelio proliferante – mutazioni in APC

• Cromosoma 5
• Le cellule epiteliali sfuggono parzialmente al controllo del
ciclo cellulare
• Le cellule si dividono per formare un piccolo cluster che
porta ad un polipo
• Gli individui eterozigoti ne formano da 100 a 1000

2. Adenoma intermedio- Mutazioni dell’oncogene ras

• Cromosoma 12
• Crescita maggiore e protrusioni a forma di dita

3. Adenoma avanzato – DCC/18q - deleted in colon cancer

4. Adenocarcinoma - Perdita o inattivazione di TP53/17p13
MODELLO “MULTI-HIT”
 HNPCC: CARCINOMA DEL COLON.-RETTO FAMILIARE NON
POLIPOSICO

• Germline mutation in DNA mismatch repair genes resulting in

microsatellite instability
• Inherited in AD manner
• Accounts for 1-3% of all cases of CRC (based on detection of
germline mutations)

GENI DI RISPOSTA AL DANNO DEL DNA

Riparano i danni del DNA

La perdita della loro funzione determina l’accumulo di mutazioni in altri geni cruciali

Considerati un gruppo di GENI SOPPRESSORI

HNPCC
 Microsatellite Instability Pathway

• Various repair mechanisms are available to correct any errors occurring

during DNA replication
• One type of error called “slippage” can occur during the replication of
microsatellite sequences by DNA polymerase
• Microsatellite DNA sequences are defined as short dinucleotide or
mononucleotide repeats
• These sequences are usually within non coding regions although some
genes contain microsatellites within coding regions (e.g., TGF-ß receptor
II, Insulin like growth factor II receptor, regulators of the cell cycle e.g.
E2F4, regulators of apoptosis e.g. BAX and even the MMR genes
themselves)
 Early diagnosis of hereditary of colorectal cancer

APC mutations MLH1(51%), MSH6(45%)

MSH2, PMS1 and PMS2(3%) mutations

Familial adenomatous polyposis Hereditary non-polyposis colon cancer

>15: annual colonscopy >25: annual colonscopy

>25: annual upper gastrointestinal >35: annual colonscopy, CA-125, gynecologic

endoscopies examination, transvaginal ultrasonography

No specific advice for a slithly higher risk of >45: BSO (bilateral salpingo-oophorectomy)
hepatoblastoma (in childhood) and papillary preferably with LAVH (laparoscopic trans-
thyroid cancer (in young women) vaginal histerectomy)