Valutazione di EGFR con FISH
in varie malattie neoplastiche
V. Martin
12-02-2008
Formazione interna
Cos’è EGFR?
Epidermal Growth Factor Receptor
nucleo membrana citoplasmatica
• Recettore transmembrana di tipo TK.
• Codificato dal gene EGFR, che mappa in
7p12.
• Attivato attraverso il binding con lo
specifico ligando (EGF).
• Innesca pathway downstreams con
numerosi effetti tra cui:
- la proliferazione cellulare
- la capacità del tumore di invadere
- l’ angiogenesi
- la capacità del tumore di dare metastasi.
EGFR e il cancro
Nei tumori solidi EGFR è
deregolato in diversi modi:
- a livello del recettore
- a livello del gene che lo
codifica
nucleo membrana citoplasmaticaEGFR e il cancro
1) overespressione proteica
(IHC)
2) mutazione genica
(sequenziamento)
3) alterazione numero di copie
geniche (FISH)
Cellula tumorale
EGFR e il cancro
1) overespressione proteica
(IHC)
2) mutazione genica
(sequenziamento)
3) alterazione numero di copie
geniche (FISH)
EGFR e il cancro
1) overespressione proteica
(IHC)
2) mutazione genica
(sequenziamento)
3) alterazione numero di copie
EGFR FISH
normale normale normale
EGFR FISH
pattern normale (disomia)
EGFR FISH
aneusomia cromosoma 7
EGFR FISH
EGFR FISH
amplificazione gene EGFR
EGFR FISH
amplificazione gene EGFR
EGFR FISH
aneusomia cromosoma 7 & amplificazione gene EGFR
EGFR FISH
SCORING SYSTEM ICP
numero segnali gene EGFR
R =
5
4
3
2
1
n
BASSA POLISOMIA
≥
50%
3-4 copie bilanciate gene
EGFR-centromero 7
ALTA POLISOMIA
≥
50%
> 4 copie bilanciate gene
EGFR-centromero 7
AMPLIFICAZIONE GENICA
≥
10%
Rapporto (R) gene
EGFR/centromero 7
≥
3
DISOMIA/NORMALE
≥
50%
2 copie bilanciate gene
EGFR-centromero 7
LOSS/PERDITA
≥
50%
1 copia bilanciata
gene EGFR-centromero 7
FISH
PATTERN
CATEGORIA
FREQUENZA
ANOMALIA
EGFR FISH
CRITERI DI CLASSIFICAZIONE ICP
EGFR FISH
in
tumori solidi
Importanza di EGFR
)
Valore prognostico:
correla con progressione
tumorale e scarsa
sopravvivenza
Cellula tumorale
MoAb
TKI
)
Valore predittivo:
- anticorpi monoclonali (MoAb)
- inibitori tirosi kinasici (TKI)
CARCINOMI COLORETTALI (CRC)
--1/43 (2%) 10/43 (23%) 7/43 (16%) 16/43 (37%) 9/43 (21%) 1) Loss if 1 copy of chr 7 in >50% of cells2) Disomy if 2 copies of chr 7 in >50% of cells 3) Low polysomy If 3 or 4 copies of chr 7 in >50% of cells 4) Marked polysomy if >4 copies of chr 7 in >50% of cells 5) Amplification if R> 3 in at least 10% of cells mCRC
43 Our unpublished data
Patients whit amplification or marked polysomy have a increased likelihood to responde to cetuximab therapy (depending from K-ras and PTEN status)while the disomic one in generally are resistant 0/27 (0%)
3/27 (11%) 0/27 (0%) 16/27 (59%) 8/27 (30%) 1) Loss if 1 copy of chr 7 in >50% of cells
2) Disomy if 2 copies of chr 7 in >50% of cells 3) Low polysomy If 3 or 4 copies of chr 7 in >50% of cells 4) Marked polysomy if >4 copies of chr 7 in >50% of cells 5) Amplification if R> 3 in at least 10% of cells mCRC
27 Frattini et al. 2007
Patients with EGFR CNG have an increased likelihoood to responde to cetuximab therapy 43/85 (50%)
42/85 (50%) Score EGFR /nucleus and use the cut off value.
FISH + if > 2.92 FISH - if <= 2.92 mCRC 85 Cappuzzo et al. 2007
Patients with disomic or low polisomy of chr7 have a reduced likelihoood to responde to panitumumab
38-39/58 20-19/58 Score EGFR gene/nucleus and use the cut off value. FISH + if > 2.5 and/or > 40% chr 7 polysomy FISH - if <= 2.5 and/or <= 40% chr 7 polysomy mCRC
58 Sartore-Bianchi et al 2007
Of the 9 patients with CNG 8 respondered and 1non responded to cetuximab or panitumumab,suggesting a genetic basis of response to antiEGFR treatment 9/20 (45%)
Score EGFR gene/ nucleus.
Increased EGFR CNG was defined as the presence of three or more signals per nucleus.
mCRC 31 Moroni et al. 2005 --37/48 (77%) 4/48 (8%) 7/48 (15%) 1) Balanced if R > 0.8 but <1.2 2) Copy loss if R <0.8 3) Copy gain if R ≥1.2 RC 48 Sauer et al. 2005 --11/244 (4%)
Amplification when a definite cluster or more than 10 orange signals were found.
CRC 244 Ooi et al. 2004 Observations Number of cases and % FISH interpretation criteria
Type Cases Authors and year --1/43 (2%) 10/43 (23%) 7/43 (16%) 16/43 (37%) 9/43 (21%) 1) Loss if 1 copy of chr 7 in >50% of cells
2) Disomy if 2 copies of chr 7 in >50% of cells 3) Low polysomy If 3 or 4 copies of chr 7 in >50% of cells 4) Marked polysomy if >4 copies of chr 7 in >50% of cells 5) Amplification if R> 3 in at least 10% of cells mCRC
43 Our unpublished data
Patients whit amplification or marked polysomy have a increased likelihood to responde to cetuximab therapy (depending from K-ras and PTEN status)while the disomic one in generally are resistant 0/27 (0%)
3/27 (11%) 0/27 (0%) 16/27 (59%) 8/27 (30%) 1) Loss if 1 copy of chr 7 in >50% of cells
2) Disomy if 2 copies of chr 7 in >50% of cells 3) Low polysomy If 3 or 4 copies of chr 7 in >50% of cells 4) Marked polysomy if >4 copies of chr 7 in >50% of cells 5) Amplification if R> 3 in at least 10% of cells mCRC
27 Frattini et al. 2007
Patients with EGFR CNG have an increased likelihoood to responde to cetuximab therapy 43/85 (50%)
42/85 (50%) Score EGFR /nucleus and use the cut off value.
FISH + if > 2.92 FISH - if <= 2.92 mCRC 85 Cappuzzo et al. 2007
Patients with disomic or low polisomy of chr7 have a reduced likelihoood to responde to panitumumab
38-39/58 20-19/58 Score EGFR gene/nucleus and use the cut off value. FISH + if > 2.5 and/or > 40% chr 7 polysomy FISH - if <= 2.5 and/or <= 40% chr 7 polysomy mCRC
58 Sartore-Bianchi et al 2007
Of the 9 patients with CNG 8 respondered and 1non responded to cetuximab or panitumumab,suggesting a genetic basis of response to antiEGFR treatment 9/20 (45%)
Score EGFR gene/ nucleus.
Increased EGFR CNG was defined as the presence of three or more signals per nucleus.
mCRC 31 Moroni et al. 2005 --37/48 (77%) 4/48 (8%) 7/48 (15%) 1) Balanced if R > 0.8 but <1.2 2) Copy loss if R <0.8 3) Copy gain if R ≥1.2 RC 48 Sauer et al. 2005 --11/244 (4%)
Amplification when a definite cluster or more than 10 orange signals were found.
CRC 244 Ooi et al. 2004 Observations Number of cases and % FISH interpretation criteria
Type Cases Authors and year
R= ratio EGFR gene signals/CEP signals CNG: copy number gain
)
Pazienti con
amplificazione o alta
polisomia di EGFR
possono beneficiare del
farmaco mirato
(cetuximab o
panitumumab)
)
Pazienti disomici o
con bassa polisomia
sono resistenti
Famaco anti-EGFR (cetuximab) FDA approved
2
12
3
NR
6
4
0
PR
A
HP
D
Risposta
clinica a
cetuximab
FISH EGFR
27 mCRC
British Journal of Cancer 2007;97:1139-1145
EGFR-FISH
CARCINOMA COLORETTALE
-ICP-TUMORI DEL POLMONE
NON A PICCOLE CELLULE (NSCLC)
After gefitinib treatment, EGFR FISH + patients had a significant improvement in response, time to progression and survival with respect to EGFR FISH- patients.. 25 (69%)
11 (31%) FISH +:
≥40% of cells displaying >= 4 copies of the EGFR signals or with gene amplification**
FISH -:
<than 40% of cells displaying >= 4 copies of the EGFR gene and no gene amplification**
NSCLC 36 Cappuzzo et al 2007
Increased EGFR gene copy number is associated with improved survival after gefitinib therapy.
26 (32%)
55 (68%) FISH +:
>= 40% of cells displaying >= 4 copies of the EGFR signals or with gene amplifications**
FISH -:
<than 40% of cells displaying >= 4 copies of the EGFR gene and no gene amplification** NSCLC (BAC subtype) 81 Hirsh et al 2005
Increased EGFR copy number were associated with responsiveness to erlotinib but not with increased survival. 56 (45%) high polysomy and amplified 1) disomy 2) low trisomy 3) high trisomy 4) low polysomy 5) high polysomy 6) amplification* NSCLC 125 Tsao et al 2005
After gefitinib treatment, EGFR FISH+ patients had a significant improvement in response, time to progression and survival with respect to EGFR FISH- patients.
33 (33%) 69 (67%) 1) disomy 2) low trisomy 3) high trisomy 4) low polysomy 5) high polysomy 6) amplification
Similar outcome of patients with high gene copy number suggested a combination in two classes: FISH +: amplification and/or high polysomy FISH -: disomy and/or low polysomy NSCLC
102 Cappuzzo et al 2005
Patients whit high gene copy number trend toward poor prognosis. 73 (40%) 70 (38%) 23 (13%) 17 (9%) 1) disomy 2) trisomy 3) polysomy 4) amplification*
low level amplification if R2.1>R>3.0 high level amplification if R >= 3 NSCLC 183 (TMA) Hirsh et al 2003 Observations Number of cases and % FISH interpretation criteria
Type Cases Authors and year
After gefitinib treatment, EGFR FISH + patients had a significant improvement in response, time to progression and survival with respect to EGFR FISH- patients.. 25 (69%)
11 (31%) FISH +:
≥40% of cells displaying >= 4 copies of the EGFR signals or with gene amplification**
FISH -:
<than 40% of cells displaying >= 4 copies of the EGFR gene and no gene amplification**
NSCLC 36 Cappuzzo et al 2007
Increased EGFR gene copy number is associated with improved survival after gefitinib therapy.
26 (32%)
55 (68%) FISH +:
>= 40% of cells displaying >= 4 copies of the EGFR signals or with gene amplifications**
FISH -:
<than 40% of cells displaying >= 4 copies of the EGFR gene and no gene amplification** NSCLC (BAC subtype) 81 Hirsh et al 2005
Increased EGFR copy number were associated with responsiveness to erlotinib but not with increased survival. 56 (45%) high polysomy and amplified 1) disomy 2) low trisomy 3) high trisomy 4) low polysomy 5) high polysomy 6) amplification* NSCLC 125 Tsao et al 2005
After gefitinib treatment, EGFR FISH+ patients had a significant improvement in response, time to progression and survival with respect to EGFR FISH- patients.
33 (33%) 69 (67%) 1) disomy 2) low trisomy 3) high trisomy 4) low polysomy 5) high polysomy 6) amplification
Similar outcome of patients with high gene copy number suggested a combination in two classes: FISH +: amplification and/or high polysomy FISH -: disomy and/or low polysomy NSCLC
102 Cappuzzo et al 2005
Patients whit high gene copy number trend toward poor prognosis. 73 (40%) 70 (38%) 23 (13%) 17 (9%) 1) disomy 2) trisomy 3) polysomy 4) amplification*
low level amplification if R2.1>R>3.0 high level amplification if R >= 3 NSCLC 183 (TMA) Hirsh et al 2003 Observations Number of cases and % FISH interpretation criteria
Type Cases Authors and year
TMA=tissue microarray R= ratio EGFR gene signals/CEP signals Amplification*= definied as clustered unbalanced gain of the EGFR gene Amplification**=defined by the presence of tight gene clusters, or R>=2, or >=15 copies of the gene per cell in >= 10% analyzed cell
Farmaco anti-EGFR (gefitinib)
)
Pazienti trattati con
gefitinib o erlotinib che
presentano
amplificazione genica
o alta polisomia di
EGFR hanno maggiori
possibilità di risposta e
migliore sopravvivenza
rispetto a quelli con
disomia o bassa
polisomia di EGFR.
TUMORI SQUAMOCELLULARI
TESTA COLLO (HNSCC)
Patients whose tumors had aberrant copy number were more advanced and had a poorer clinical outcome. 32/134 abnormal EGFR GCN : 22 con increased and 10 with decreased 102 disomic FISH +:
>= 40% of cells displaying >= 4 copies of the EGFR signals or with gene amplification**
FISH -:
<than 40% of cells displaying >= 4 copies of the EGFR gene and no gene amplification**
HNSCC 134 Temam et al 2007
Patients with FISH+ tumours had a worse survival.
43 (50%) 42 (50%) FISH + : amplification and/or high polysomy
FISH -: disomy and/or low polysomy HNSCC
86 Chung et al. 2006
Patients whose tumors had either gene amplification or deletion had a poorer survival
(only abstract available) 14.24% amplification 6.10 deletion HNSCC of larynx 59 Morrison et al 2005 --(only abstract available)
23 aneusomy 7 amplified HNSCC 33 Marholova et al .2005 --2 (0.--2%) 112 (10%) 1) “normal”: samples that did not met the criteria for gain or amplification
2) “gain”: R> 1.5 and <4 in at least 10% of cells 3) amplification: R>4 or tight clusters in 10% of cells HNSCC (of lariynx) 1080 (TMA) Koyonova et al. 2005
No correlation with survival. 79 (13%)
Amplification when >10% of cells showing more than 8 signals or tight cluster. HNSCC 609 (TMA) Freier et al. 2003 Observations Number of cases and % FISH interpretation criteria
Type Cases Authors and year
Patients whose tumors had aberrant copy number were more advanced and had a poorer clinical outcome. 32/134 abnormal EGFR GCN : 22 con increased and 10 with decreased 102 disomic FISH +:
>= 40% of cells displaying >= 4 copies of the EGFR signals or with gene amplification**
FISH -:
<than 40% of cells displaying >= 4 copies of the EGFR gene and no gene amplification**
HNSCC 134 Temam et al 2007
Patients with FISH+ tumours had a worse survival.
43 (50%) 42 (50%) FISH + : amplification and/or high polysomy
FISH -: disomy and/or low polysomy HNSCC
86 Chung et al. 2006
Patients whose tumors had either gene amplification or deletion had a poorer survival
(only abstract available) 14.24% amplification 6.10 deletion HNSCC of larynx 59 Morrison et al 2005 --(only abstract available)
23 aneusomy 7 amplified HNSCC 33 Marholova et al .2005 --2 (0.--2%) 112 (10%) 1) “normal”: samples that did not met the criteria for gain or amplification
2) “gain”: R> 1.5 and <4 in at least 10% of cells 3) amplification: R>4 or tight clusters in 10% of cells HNSCC (of lariynx) 1080 (TMA) Koyonova et al. 2005
No correlation with survival. 79 (13%)
Amplification when >10% of cells showing more than 8 signals or tight cluster. HNSCC 609 (TMA) Freier et al. 2003 Observations Number of cases and % FISH interpretation criteria
Type Cases Authors and year
R= ratio EGFR gene signals/CEP signals
Amplification*= definied as clustered unbalanced gain of the EGFR gene
Amplification**=defined by the presence of tight gene clusters, or R>=2, or >=15 copies of the gene per cell in >= 10% analyzed cell
Famaco anti-EGFR (cetuximab) FDA approved
)
Pazienti con
alterazione del numero
di copie geniche di
EGFR hanno prognosi
sfavorevole
EGFR amplification confers shorter survival, unfavourable prognostic marker.
46/114 (40%)
67°/114(59%) 1) Amplification when R>2 in >10% of tumor cells or
innumerable clusters of EGFR signals. 2) Polysomy when >10% of nuclei containing >=3 of chr7 High grade small-cell glioma s 114 Korshunov et al 2004
EGFR amplification is a strong indicator of adverse outcome for “young adults”(pts <50 years). 60/189 (31%)
171°/189 (90%) 1) Amplification when R>2 in >10% of tumor cells or innumerable clusters of EGFR signals. 2) Polysomy when >20% of nuclei containing >=3 of chr7 GBM (age <50 years) 189 Korshunov.et al 2004
EGFR amplification is an independent predictor of prolonged survival in patients with GBM who were elder than 60 years of age. AA:
11/63 (17%) GBM: 46/111 (41%) Amplification if R>=1.2 in > 10% of tumor cells or > 3 EGFR signals. 63 AA 111 GBM 174 Smith et al. 2001 Observations Number of cases and % FISH interpretation criteria
Type Cases Authors and
year
EGFR amplification confers shorter survival, unfavourable prognostic marker.
46/114 (40%)
67°/114(59%) 1) Amplification when R>2 in >10% of tumor cells or
innumerable clusters of EGFR signals. 2) Polysomy when >10% of nuclei containing >=3 of chr7 High grade small-cell glioma s 114 Korshunov et al 2004
EGFR amplification is a strong indicator of adverse outcome for “young adults”(pts <50 years). 60/189 (31%)
171°/189 (90%) 1) Amplification when R>2 in >10% of tumor cells or innumerable clusters of EGFR signals. 2) Polysomy when >20% of nuclei containing >=3 of chr7 GBM (age <50 years) 189 Korshunov.et al 2004
EGFR amplification is an independent predictor of prolonged survival in patients with GBM who were elder than 60 years of age. AA:
11/63 (17%) GBM: 46/111 (41%) Amplification if R>=1.2 in > 10% of tumor cells or > 3 EGFR signals. 63 AA 111 GBM 174 Smith et al. 2001 Observations Number of cases and % FISH interpretation criteria
Type Cases Authors and
year
GLIOBLASTOMI (GBM)
AA: anaplastico astrocytoma R= ratio EGFR gene signals/CEP signals
° polysomy of chromosome 7 concomitant to EGFR gene amplification
)
Pazienti con
amplificazione di
EGFR hanno
prognosi
sfavorevole
Amplificazione di EGFR è marcatore diagnostico di GBM primario
TUMORI DELLA MAMMELLA (BC)
R= ratio EGFR gene signals/CEP signals °= pac probe dig-labelled for EGFR gene high grade ductal carcinoma with myoepithelial differentiation (DCMD) locally advanced breast cancer (LABC)
basal like breast cancer (BLBC) methaplastic breast cancer (MBC)
--0/18 (0%) 7/18 (39%) 0/18 (0%) 6/18 (33%) 5/18 (28%) 1) Loss if 1 copy of chr 7 in >50% of cells
2) Disomy if 2 copies of chr 7 in >50% of cells 3) Low polysomy If 3 or 4 copies of chr 7 in >50% of cells 4) Marked polysomy if >4 copies of chr 7 in >50% of cells 5) Amplification if R> 3 in at least 10% of cells BLBC 20 Our unpublished data --0/29(0%) --BLBC +MBC 29 Schiller et al. poster 2006 --0/48 (0%) --LABC 48 Corzo et al. 2005 --0/24 (0%) Amplification if R>2.0 DCMD 24 Shien et al. 2006 --6/57 (11%) Amplification if >than 4 signals per cell are present.
-Criteria of Steodl et al 2002 (urinary bladder tumor)-Phylloid tumours 58° (TMA) Kersting et al 2006
EGFR gene amplification is a rare event in invasive breast cancer. 8/170 (4.7%)
Amplification if >than 4 signals per cell are present -Criteria of Steodl et al 2002 (urinary bladder tumor)-IDC 222° (TMA) Kersting et al. 2004 Observations Number of cases and % FISH interpretation criteria
Type Cases Authors and year --0/18 (0%) 7/18 (39%) 0/18 (0%) 6/18 (33%) 5/18 (28%) 1) Loss if 1 copy of chr 7 in >50% of cells
2) Disomy if 2 copies of chr 7 in >50% of cells 3) Low polysomy If 3 or 4 copies of chr 7 in >50% of cells 4) Marked polysomy if >4 copies of chr 7 in >50% of cells 5) Amplification if R> 3 in at least 10% of cells BLBC 20 Our unpublished data --0/29(0%) --BLBC +MBC 29 Schiller et al. poster 2006 --0/48 (0%) --LABC 48 Corzo et al. 2005 --0/24 (0%) Amplification if R>2.0 DCMD 24 Shien et al. 2006 --6/57 (11%) Amplification if >than 4 signals per cell are present.
-Criteria of Steodl et al 2002 (urinary bladder tumor)-Phylloid tumours 58° (TMA) Kersting et al 2006
EGFR gene amplification is a rare event in invasive breast cancer. 8/170 (4.7%)
Amplification if >than 4 signals per cell are present -Criteria of Steodl et al 2002 (urinary bladder tumor)-IDC 222° (TMA) Kersting et al. 2004 Observations Number of cases and % FISH interpretation criteria
Type Cases Authors and year 18
CONCLUSIONI
EGFR
≠
HER2 !!!
EGFR FISH è
un’analisi complessa …
