Dal GIST al K-RAS
OVVERO
Dall’ E.E. al D.N.A.
Come è cambiata e sta cambiando
l’Anatomia Patologica
Gallarate Aprile 2009
Imatinib mesylate
(also called Gleevec® or STI571)
is approved by the U.S. Food and Drug
Administration (FDA) for the treatment
of some forms of adult and pediatric chronic
myelogenous leukemia (CML), and for the
treatment of a rare form of cancer called
gastrointestinal stromal tumor (GIST).
STI571
Foto cd117
GLEEVEC
foto
Foto cml
Foto gist
MIB1
Alfa-actina
Alfa-actina
CD117
GastroIntestinal Stromal Tumors
Acivating mutations in c-KIT and PDGFRA
5%
About 80-90% of patients show mutations *
Antonescu RC, Clin Cancer Res 2005; 11: 4182-90
GastroIntestinal Stromal Tumors
Acivating mutations in c-KIT and PDGFRA
About 75% of GISTs shows mutations on exon 11 and are
related to Imatinib sensitivity (>80% clinical response)
About 10%- 15% of GISTs shows mutations on exon 9
and have an aggressive clinical behaviour
Fletcher JA and Rubin BP ; Current Opinion in Genetics & Development, 2007; 17: 3-7
GastroIntestinal Stromal Tumors
Imatinib or Sunitinib Resistance
Mutations in exon 9 are generally associated to primary resistance
Mutations in exon 13, 14 and 17 of c-KIT gene are subsequent to
exon 11 mutations and are generally associated to acquired resistance
Antonescu RC, Clin Cancer Res 2005; 11: 4182-90
Heinrich MC, J.Clin. Oncol. 2006; 24: 4764-74
Mutazioni iniziali e secondarie
Mutazioni iniziali
Exon 9
Resistenza
primaria
Exon 11
Mutazioni secondarie
Exon 13
Exon 17
GLEEVEC
Membrana
cellulare
Resistenza
secondaria
CD117
Dr. Lei Chen and others at M.D. Anderson Cancer Center in
Houston have identified a secondary mutation that occurs in
KIT kinase domain 1 (exon 13). This secondary mutation
correlates with resistance to Gleevec.
Among the 130 patients in the M.D. Anderson study, 12 who
had an excellent initial response were chosen for further study.
Seven of these patients originally had exon 11 mutations and
five had exon 9 mutations. Five of these patients developed
resistance in a total of six tumors. In each case, in addition to
the original exon 11 or exon 9 mutation, a new secondary
exon 13 mutation, Val654Ala, was identified. In each of these
cases, the secondary mutation was identical and the resistant
tumors now contained both the primary mutation
(exon 11 or exon 9) and the new exon 13 mutation.
In the seven patients who did not develop resistance no
secondary mutations were found.
Dalla diagnosi di
LEIOMIOBLASTOMA
a quella di GIST CD117 +
Parametri prognostici
Valutazione mutazioni
esoni
9 11
13 17
GIST is a rare form of cancer
BUT
LUNG ?
COLON ?
TAILOR AIFA
TArceva Italian Lung Optimization tRial
Lo studio deve valutare se i casi di
K polmonare non mutati per EGFR
siano responsivi al Tarceva dando
per acquisito che i mutati lo siano
• Siamo sicuri che tutti casi ( o almeno gli
adenoK giovanili ) siano studiati per stato
mutazionale di EGFR ?
• Quanti fanno sistematicamente una
valutazione dello stato mutazionale di
EGFR nel K polmonare ed in quali casi ?
La famiglia HER (erbB) ed i suoi ligandi
EGF
TGFa
Amphiregulin
b-cellulin
HB-EGF
Epiregulin
Tyrosine kinase
domain
Heregulins
NRG2
NRG3
Heregulins
b-cellulin
100
44
36
48
100
82
59
79
100
33
24
28
ErbB-1
Her1
EGFR
ErbB-2
Her2
neu
ErbB-3
Her3
Cysteine-rich
domains
C-terminus
ErbB-4
Her4
Location of mutations in TK domain of EGFR gene
del ELREA (E746-A750)
Mechanisms of resistance to TK inhibitors
Exon 19 del E745-A750
Exon 20 T790M
PARLIAMO DI K
COLON E DI K-RAS
CANCRO COLON-RETTO
90 CASI x 105 ANNUI
IN LOMBARDIA 8000 CASI ANNUI
RICADUTA CIRCA 4000 Pz
La famiglia HER (erbB) ed i suoi ligandi
CETUXIMAB
Tyrosine kinase
domain
HERCEPTIN
EGF
TGFa
Amphiregulin
b-cellulin
HB-EGF
Epiregulin
Heregulins
NRG2
NRG3
Heregulins
b-cellulin
100
44
36
48
100
82
59
79
100
33
24
28
ErbB-1
Her1
EGFR
ErbB-2
Her2
neu
ErbB-3
Her3
Cysteine-rich
domains
C-terminus
ErbB-4
Her4
NESSUN RISULTATO
ATTENDIBILE
• DIFFICOLTA’ TECNICHE DI RECUPERO
ANTIGENICO
• NON RESPONSIVITA’ CASI POSITIVI
ALLA IIC
• RESPONSIVITA’ CASI NEGATIVI ALLA
IIC
EGFR
Centr
COLON NORMALE
CARCINOMA -- POLISOMIA
2 green 2 red
4 green 4 red
Perdita materiale nucleare
2 green 2 red
CELLULA TUMORALE
CELLULA NORMALE
CARCINOMA – AMPLIFICAZIONE
CARCINOMA – AMPLIFICAZIONE
APPROCCIO AUTOMATICO DI LETTURA
Gene copy number for epidermal growth
factor receptor (EGFR) and clinical
response to antiEGFR treatment in
colorectal cancer: a cohort study
M.Moroni, S.Veronese,S.Benvenuti,
G.Marrapese,A. Sartore-Bianchi,
F.Dinicolantonia,M.Gambacorta,S.Siena,
A.Bardelli.
Lancet Oncology 2005
EGFR gene copy number analysis
Disomy
EGFR/CEP 7
Polysomy
> 2,50 copies per cell
Focal amplification
> 40% polysomic cells with
> 3 EGFR copies
PFS and OS according to the proposed cut off values
Sartore-Bianchi A et al. J Clin Oncol 2007
Clinical usefulness of EGFR gene copy
number as a predictive marker in
colorectal cancer patients treated with
Cetuximab : A FISH study
Personeni N. et Al
Clin Cancer Res 2008
E’ in corso uno studio pluricentrico per
standardizzazione tecnica e interpretazione
risultati F.I.S.H. Her1 su sezioni
SEQUENZA E G F R :
ASSENZA DI MUTAZIONI
ESONI 18 , 19 , 20 , 21
TK signaling cascade
Mutated KRAS is prevalent in
many different tumor types
Cancer Type
Reported Incidence
of Mutated KRAS
Pancreatic
72 – 90%
Colon
32 – 57%
Lung
15 – 50%
Ovarian
5 – 50%
Gall bladder
14 – 38%
Multiple myeloma
16 – 33%
Adapted from: Friday BB and Adjei AA. Biochim. Biophys. Acta. 2005; 1756:127-144
K-RAS Mutations
WT
……
GGA GCT GGT GGC GTA GGC ……
Wild-type
G12S
G12D
G12D
G12A
G12V
G12S
G12R
G12C
G13D
Gly12Asp
Gly12Ala
Gly12Val GGT>GTT
Gly12Ser
Gly12Arg
Gly12Cys
Gly13Asp
GGT>GAT
GGT>GCT
GGT>AGT
GGT>CGT
GGT>TGT
GGC>GAC
2008 ASCO Annual Meeting
Gastrointestinal (Colorectal) Cancer
KRAS status and efficacy in the first-line treatment of patients with mCRC
treated with FOLFIRI with or without Cetuximab : the CRYSTAL experience
Van Cutsem E et al; JCO 26; 2008 (May 20 Suppl; abstr 2)
540 archived tumor material
WT Patients (64.4%)
Progression Free survival (PFS)
p=0.0167
Overall response
p=0.0025
(OS)
KRAS mutated Patients (35.6%)
Progression Free survival (PFS)
p=0.75
Overall response in (OS)
p=0.46
Conclusion : KRAS mutation status has a predictive value for treatment
with FOLFIRI + Cetuximab in the first-line treatment of mCRC
2008 ASCO Annual Meeting
Gastrointestinal (Colorectal) Cancer
KRAS status and efficacy of first-line treatment of patients with mCRC with
FOLFOX with or without Cetuximab : the OPUS experience
Bokemeyer C et al; JCO 26; 2008 (May 20 Suppl; abstr 4000)
233 tumor samples
99/233 (42.5%) KRAS mutated
134/233 (57.5%) KRAS WT
PFS and overall Response Rate (RR) by KRAS mutation status
KRAS status
Median PFS Median PFS
Overall RR
Overall RR
(mo)
(%)
(%)
(mo)
Cetuximab +
FOLFOX
FOLFOX
Cetuximab +
FOLFOX
FOLFOX
Wild-type
7.7 (n=61)
7.2 (n=73)
HR = 0.57
p=0.02
61 (n=61)
37 (n=73)
p=0.01
Mutation
5.5 (n=52)
8.6 (n=47)
HR = 1.83
p=0.02
33 (n=52)
49 (n=47)
p=0.11
HR : Hazard Ratio
Conclusions: The benefit from addiction of Cetuximab to standard treatment is
higher for KRAS WT population.
Best
response
Time To Progression according
KRas and BRaf mutational status
Ras/Raf
mutations
+
Total
SD+PD
15
21
(58.3%)
36
PR
10
1
(9.0%)
11
Total
25
22
47
p =0.005 (Fisher's exact test)
Logistic regression
Odds Ratio = 0.071 (CI95%=(0.008, 0.619); p=0.017)
Constitutive activation of EGFR effectors
MoAb
RAS
RAF
Protein kinase BRaf mutations
PTEN inactivation or loss of expression
(Phosphatase and Tensin homolog deleted on chromosome 10)
MoAb
pAkt
KRAS
12p12.1
v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
The KRAS gene is located on the short (p) arm of chromosome 12 at position 12.1.
More precisely, the KRAS gene is located from base pair 25,249,446 to base pair
25,295,120 on chromosome 12.
1
2
3
4
5
6
gene
ENSG00000133703
Exons: 6
Transcript length: 5,419 bps
Protein length: 189 residues
KRAS gene
5' upstream sequence
…………………............cccggccccgaactcatcggtgtgctcggagctcgattttcctaggcggc
Exon 1
(170)GGCCGCGGCGGCGGAGGCAGCAGCGGCGGCGGCAGTGGCGGCGGCGAAGGTGGCG
GCGGCTCGGCCAGTACTCCCGGCCCCCGCCATTTCGGACTGGGAGCGAGCGCGGCGCAGG
CACTGAAGGCGGCGGCGGGGCCAGAGGCTCAGCGGCTCCCAGGTGCGGGAGAGAG
Intron 1-2
(5355) gtacggagcg........................................ttattataag
Exon 2
(122)GCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGG
CAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAA
CAATAGAG
Intron 2-3
(17.861) gtaaatcttg........................................cccttctcag
Exon 3
(179)GATTCCTACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTC
GACACAGCAGGTCAAGAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGG
AGGGCTTTCTTTGTGTATTTGCCATAAATAATACTAAATCATTTGAAGATATTCACCATTATAG
Intron 3-4
(1.460) gtgggtttaa........................................tctttcccag
Exon 4
(160)AGAACAAATTAAAAGAGTTAAGGACTCTGAAGATGTACCTATGGTCCTAGTAGGA
AATAAATGTGATTTGCCTTCTAGAACAGTAGACACAAAACAGGCTCAGGACTTAGCAAGA
AGTTATGGAATTCCTTTTATTGAAACATCAGCAAAGACAAGACAG
Intron 4-5
(10.053) gtaagtaaca........................................tacaatgcag
Exon 5
(124)AGAGTGGAGGATGCTTTTTATACATTGGTGAGGGAGATCCGACAATACAGATTGA
AAAAAATCAGCAAAGAAGAAAAGACTCCTGGCTGTGTGAAAATTAAAAAATGCATTATAA
TGTAATCTG
Intron 5-6
(5.525) gtaagtttaa........................................tgtatttcag
Exon 6
(4.664)GGTGTTGATGATGCCTTCTATACATTAGTTCGAGAAATTCGAAAACATAAAGAAAAGATG
………………………………………………………………………………………………………………………
3' downstream sequence
actatgagtgtgtatttattcatgaaatttgaactgtttgccccgaaatg................................................
KRAS mutations in adenocarcinomas of large intestine
Gene Name
Sample Number
Positive
Samples
Percent
Mutated
KRAS
2657
995
37%
APC
937
365
38%
BRAF
824
114
13%
CTNNB1
894
51
5%
PIK3CA
254
41
16%
COSMIC
Wellcome Trust SANGER Institute
K-RAS Mutations
Electropherograms
……
GGA GCT GGT GGC GTA GGC ……
Wild-type
SnaPShot
WT
Sequencing
G12S
G12D
G12D
KRAS Testing
Characteristics of Selected Assays
Methodology
Direct sequencing
Allele-specific
real-time PCR
Kit
(Manufacturer)
Mutations
detected
Individual
reference
laboratory
All
TaqManTM KRASspecific primer +
probe sets
• Direct sequence information
• Independent of allele present
• Result depends on individual
Any designed
(Applied Biosystems)
Real-time PCR
detection using
allele-specific
primer-probes
Allele-specific
ELISA microplate
DNA hybridisation
www.appliedbiosystems.com
www.invitek.eu
TheraScreenTM
K-RAS Kit
(DxS)
Invigene®
K-ras
Genotyping Kits
(Invitek)
Features
G12D
G12A
G12V
G12S
G12R
G12C
G13D
G12S
G12R
G12C
G12D
G12A
G12V
G13S
G13R
G13C
G13D
G13A
G13V
www.dxsgenotyping.com
www.qiagen.com
assay design
• Single-tube amplification +
detection
• High sensitivity and specificity
• Single-tube amplification +
detection
• First CE-labelled kit
• Includes sample preparation
module
• Detection includes manual steps
KRAS Mutation Testing

Histopathological evaluation is essential
1.
Paraffin-embedded, formalin-fixed tissue
(biopsies, surgical samples)
2.

Macro/microdissection can enhance sensitivity
Various test methods available, all adequate
1.
Direct sequencing
- Sensitivity of standard sequencing techniques is sufficient in most cases
- Sequencing provides direct information about the type of mutation
- Detects all mutations in region sequenced
2.
Allele-specific amplification and detection
- Provides high sensitivity
- Appropriate for high-throughput or multiplex applications
- Only pre-defined mutations can be detected

Detection can be performed both on primary tumor and
metastasis
DISSEZIONE
MICROSCOPICA
CETUXIMAB SI USA IN
CASI IV° STADIO RAS W. T.
ora
• Circa il 40 % di tutti i casi di K del
grosso intestino sono o diventano IV°
stadio
• Ma circa il 50 % sono stadio II°
e su questi ?
18q allelic loss in CRC
 The short arm of the chromosome 17 (17p) and the
long arm of the chromosome 18 (18q) are frequently
loss in CRC
 Inactivation of the p53 gene (on 17p) and DCC gene
(on 18q) probably contributes to neoplastic
transformation
 Distant metastasis of CRC is associated with deletions
of 17p and 18q, and loss of 17p and 18q has prognostic
value
18q deletion and outcome in untreated CRC
18q+
18q-
The status of chromosome 18q has strong prognostic value in patients with stage
II colorectal cancer. The prognosis in patients with stage II cancer and chromosome
18q allelic loss is similar to that in patients with stage III cancer, who are thought to
benefit from adjuvant therapy. In contrast, patients with stage II disease who do not
have chromosome 18q allelic loss in their tumor have a survival rate similar to that of
patients with stage I disease and may not require additional therapy.
Jen et al. NEJM 1994
EGFR-targeted therapies
1
4
2
3
Molecular conditions for anti-EGFR efficiency
at the receptor level
at the intracellular
level
II. No downstream
oncogenic
activation:
KRAS (40%)
BRAF (10%)
PTEN (loss, 30%)
PIK3CA? (20%)
I. Activation of EGFR in the tumor:
EGFR gain of copies, ligands
overexpression
A sequential strategy:
downstream
signaling
KRAS
Cancer Res 2007
BRAF
J Clin Oncol 2008
PTEN
PIK3CA
Cancer Res 2009
?
The role of PIK3CA/PTEN (I)
Available data of the predictive role in CRC:
 Preclinical: Jawher, Cancer Res 2008
 Clinical:
-Frattini et al. Br J Cancer 2007
-Perrone et al. Ann Oncol 2009
32 pts:
-24% KRAS mut
(p=0.03)
-10% BRAF (p=ns)
-13% PIK3CA mut
(p=ns)
-10% PTEN mut
-13% PTEN loss
(p=0.02)
Perrone F et al.
Ann Oncol 2009
110 metastatic colorectal cancers patients
treated with the EGFR-targeted monoclonal
antibodies panitumumab or cetuximab
KRAS mutational status
(evaluable n=109)
*p<0.05
(p=0.019)
Mutated KRAS
32/109 (29%)
Wild-Type KRAS
77/109 (71%)
Responders
2*
20*
Non Responders
30*
57*
PIK3CA/PTEN evaluation in wild-type KRAS tumors
(evaluable n=59)
**p<0.01 (p=0.00003)
Altered
PIK3CA/PTEN
27/59 (46%)
Normal
PIK3CA/PTEN
32/59 (54%)
Responders
1**
17**
Non Responders
26**
15**
Frequency of mutations in PIK3CA and KRAS,
and loss of PTEN protein expression
 15 (13.6%) PIK3CA mutations, both in exon 9 (4 cases) and in
exon 20 (11 cases);
 32 (29.0%) KRAS mutations, occurring at codon 12 in 23 cases
(71.9%), and at codon 13 in 8 cases (25.0%); a double point
mutation involving both codons was detected in 1 case (3.1%)
 Concomitant PIK3CA and KRAS mutations were observed in 2
samples
 PTEN protein assessment was performed by
immunohistochemistry analysis. Among the 81 evaluated tumor
specimens, 32 (39.5%) showed loss of PTEN protein
168 pts
BRAF mutation predict for poor prognosis regardless the line of treatment
Molecular determinants of response
in CRC: what is reasonable to look
for in the clinical practice?
Stage II (prognostic)
experimental
18q LOH
clinical practice
Stage IV (predictive)
KRAS
BRAF
PIK3CA
PTEN
GRAZIE
Silvio Veronese : SS Patologia Molecolare
H Niguarda Milano
Andrea Sartore Bianchi : Oncologia Falk
Salvatore Siena
H Niguarda Milano
Alberto Bardelli : IRCC Candiolo