Dal GIST al K-RAS OVVERO Dall E.E. al D.N.A. Come è cambiata e sta cambiando lAnatomia Patologica...
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Transcript of Dal GIST al K-RAS OVVERO Dall E.E. al D.N.A. Come è cambiata e sta cambiando lAnatomia Patologica...
Dal GIST al K-RAS
OVVERODall’ 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).
Foto cd117
STI571
foto
GLEEVEC
Foto cml
Foto gist
MIB1
Alfa-actina
Alfa-actina
CD117
GastroIntestinal Stromal TumorsGastroIntestinal Stromal Tumors
Acivating mutations in c-KIT and PDGFRAAcivating mutations in c-KIT and PDGFRA
About 80-90% of patients show mutations *About 80-90% of patients show mutations *
Antonescu RC, Clin Cancer Res 2005; 11: 4182-90Antonescu RC, Clin Cancer Res 2005; 11: 4182-90
5%
GastroIntestinal Stromal TumorsGastroIntestinal Stromal Tumors
Acivating mutations in c-KIT and PDGFRAAcivating 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 75% of GISTs shows mutations on exon 11 and are related to Imatinib sensitivity (>80% clinical response)
Fletcher JA and Rubin BP ; Current Opinion in Genetics & Development, 2007; 17: 3-7Fletcher JA and Rubin BP ; Current Opinion in Genetics & Development, 2007; 17: 3-7
About 10%- 15% of GISTs shows mutations on exon 9and have an aggressive clinical behaviourAbout 10%- 15% of GISTs shows mutations on exon 9and have an aggressive clinical behaviour
GastroIntestinal Stromal TumorsGastroIntestinal Stromal Tumors
Imatinib or Sunitinib ResistanceImatinib or Sunitinib Resistance
Antonescu RC, Clin Cancer Res 2005; 11: 4182-90Heinrich MC, J.Clin. Oncol. 2006; 24: 4764-74Antonescu RC, Clin Cancer Res 2005; 11: 4182-90Heinrich MC, J.Clin. Oncol. 2006; 24: 4764-74
Mutations in exon 9 are generally associated to primary resistanceMutations 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 resistanceMutations in exon 13, 14 and 17 of c-KIT gene are subsequent to exon 11 mutations and are generally associated to acquired resistance
Mutazioni iniziali e secondarie
Exon 9
Membrana cellulare
Exon 11 Exon 13 Exon 17
Mutazioni iniziali Mutazioni secondarie
Resistenza secondaria
CD117
Resistenza primaria
GLEEVEC
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 whohad 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 +
Valutazione mutazioni
esoni 9 11 13 17
Parametri prognostici
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 ?
EGFTGF
Amphiregulin-cellulinHB-EGF
Epiregulin Heregulins
NRG2NRG3
Heregulins-cellulin
Cysteine-richdomains
Tyrosine kinasedomain
ErbB-1Her1
EGFR
ErbB-2Her2neu
ErbB-3Her3
ErbB-4Her4
C-terminus
100
100
100
44
82
33
36
59
24
48
79
28
La famiglia HER (erbB) ed i suoi ligandi
Location of mutations in TK domain of EGFR geneLocation of mutations in TK domain of EGFR gene
del ELREA (E746-A750)del ELREA (E746-A750)
Exon 20 T790MExon 20 T790M
Mechanisms of resistance to TK inhibitors Mechanisms of resistance to TK inhibitors
Exon 19 del E745-A750Exon 19 del E745-A750
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
EGFTGF
Amphiregulin-cellulinHB-EGF
Epiregulin Heregulins
NRG2NRG3
Heregulins-cellulin
Cysteine-richdomains
Tyrosine kinasedomain
ErbB-1Her1
EGFR
ErbB-2Her2neu
ErbB-3Her3
ErbB-4Her4
C-terminus
100
100
100
44
82
33
36
59
24
48
79
28
La famiglia HER (erbB) ed i suoi ligandi
CETUXIMAB
HERCEPTIN
NESSUN RISULTATO ATTENDIBILE
• DIFFICOLTA’ TECNICHE DI RECUPERO ANTIGENICO
• NON RESPONSIVITA’ CASI POSITIVI ALLA IIC
• RESPONSIVITA’ CASI NEGATIVI ALLA IIC
CentrEGFR
COLON NORMALE CARCINOMA -- POLISOMIA
Perdita materiale nucleare
2 green 2 red
4 green 4 red
CELLULA NORMALECELLULA TUMORALE
2 green 2 red
CARCINOMA – AMPLIFICAZIONE
CARCINOMA – AMPLIFICAZIONE
APPROCCIO AUTOMATICO DI LETTURA
Gene copy number for epidermal growthfactor 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
EGFR/CEP 7 > 2,50 copies per cell
Disomy Polysomy 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 interpretazionerisultati 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 TypeReported 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
…… GGA GCT GGT GGC GTA GGC …… …… GGA GCT GGT GGC GTA GGC ……
Wild-type
G12D
G12S
WT
G12D Gly12Asp GGT>GAT G12A Gly12Ala GGT>GCT G12V Gly12Val GGT>GTT
G12S Gly12Ser GGT>AGT G12R Gly12Arg GGT>CGT G12C Gly12Cys GGT>TGT G13D Gly13Asp GGC>GAC
2008 ASCO Annual MeetingGastrointestinal (Colorectal) Cancer
KRAS status and efficacy in the first-line treatment of patients with mCRCtreated with FOLFIRI with or without Cetuximab : the CRYSTAL experienceVan Cutsem E et al; JCO 26; 2008 (May 20 Suppl; abstr 2)
WT Patients (64.4%)
Progression Free survival (PFS) p=0.0167
Overall response (OS) p=0.0025
540 archived tumor material
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
KRAS status and efficacy of first-line treatment of patients with mCRC withFOLFOX with or without Cetuximab : the OPUS experienceBokemeyer C et al; JCO 26; 2008 (May 20 Suppl; abstr 4000)
233 tumor samples 99/233 (42.5%) KRAS mutated134/233 (57.5%) KRAS WT
PFS and overall Response Rate (RR) by KRAS mutation status
KRAS status
Median PFS(mo)Cetuximab + FOLFOX
Median PFS(mo) FOLFOX
Overall RR(%)Cetuximab + FOLFOX
Overall RR(%)
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.
2008 ASCO Annual MeetingGastrointestinal (Colorectal) Cancer
Best response
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 regressionOdds Ratio = 0.071 (CI95%=(0.008, 0.619); p=0.017)
Time To Progression according KRas and BRaf mutational status
MoAb
Protein kinase BRaf mutations
RAFRAF
Constitutive activation of EGFR effectors
RAS
PTEN inactivation or loss of expression
MoAb
pAkt
(Phosphatase and Tensin homolog deleted on chromosome 10)
v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
KRAS
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.
ENSG00000133703
Exons: 6 Transcript length: 5,419 bps Protein length: 189 residues
1 2 3 4 5 6 gene
12p12.1
5' upstream sequence …………………............cccggccccgaactcatcggtgtgctcggagctcgattttcctaggcggc
Exon 1 (170)GGCCGCGGCGGCGGAGGCAGCAGCGGCGGCGGCAGTGGCGGCGGCGAAGGTGGCGGCGGCTCGGCCAGTACTCCCGGCCCCCGCCATTTCGGACTGGGAGCGAGCGCGGCGCAGGCACTGAAGGCGGCGGCGGGGCCAGAGGCTCAGCGGCTCCCAGGTGCGGGAGAGAG
Intron 1-2 (5355) gtacggagcg........................................ttattataag
Exon 2 (122)GCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAG
Intron 2-3 (17.861) gtaaatcttg........................................cccttctcag
Exon 3 (179)GATTCCTACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGTCAAGAGGAGTACAGTGCAATGAGGGACCAGTACATGAGGACTGGGGAGGGCTTTCTTTGTGTATTTGCCATAAATAATACTAAATCATTTGAAGATATTCACCATTATAG
Intron 3-4 (1.460) gtgggtttaa........................................tctttcccag
Exon 4 (160)AGAACAAATTAAAAGAGTTAAGGACTCTGAAGATGTACCTATGGTCCTAGTAGGAAATAAATGTGATTTGCCTTCTAGAACAGTAGACACAAAACAGGCTCAGGACTTAGCAAGAAGTTATGGAATTCCTTTTATTGAAACATCAGCAAAGACAAGACAG
Intron 4-5 (10.053) gtaagtaaca........................................tacaatgcag
Exon 5 (124)AGAGTGGAGGATGCTTTTTATACATTGGTGAGGGAGATCCGACAATACAGATTGAAAAAAATCAGCAAAGAAGAAAAGACTCCTGGCTGTGTGAAAATTAAAAAATGCATTATAATGTAATCTG
Intron 5-6 (5.525) gtaagtttaa........................................tgtatttcag
Exon 6 (4.664)GGTGTTGATGATGCCTTCTATACATTAGTTCGAGAAATTCGAAAACATAAAGAAAAGATG………………………………………………………………………………………………………………………
3' downstream sequence actatgagtgtgtatttattcatgaaatttgaactgtttgccccgaaatg................................................
KRAS KRAS genegene
Gene Name Sample NumberPositive Samples
Percent Mutated
KRAS 2657 995 37%
APC 937 365 38%
BRAF 824 114 13%
CTNNB1 894 51 5%
PIK3CA 254 41 16%
COSMICWellcome Trust SANGER Institute
KRAS mutations in adenocarcinomas of large intestine
K-RAS MutationsElectropherograms
…… GGA GCT GGT GGC GTA GGC …… …… GGA GCT GGT GGC GTA GGC ……
G12D
G12S
WT
SnaPShot
Sequencing
G12D
Wild-type
KRAS Testing Characteristics of Selected Assays
MethodologyKit
(Manufacturer)Mutationsdetected Features
Direct sequencingIndividual reference laboratory
All• Direct sequence information• Independent of allele present
Allele-specific real-time PCR
TaqManTM KRAS-specific primer +
probe sets
(Applied Biosystems)
Any designed
• Result depends on individual assay design• Single-tube amplification + detection
Real-time PCR detection usingallele-specific primer-probes
TheraScreenTM
K-RAS Kit
(DxS)
G12DG12AG12VG12S
G12RG12CG13D
• High sensitivity and specificity• Single-tube amplification + detection• First CE-labelled kit
Allele-specific ELISA microplate DNA hybridisation
Invigene® K-ras Genotyping Kits
(Invitek)
G12SG12RG12CG12DG12AG12V
G13SG13RG13CG13DG13AG13V
• Includes sample preparation module• Detection includes manual steps
www.appliedbiosystems.com www.dxsgenotyping.comwww.invitek.eu www.qiagen.com
Histopathological evaluation is essential1. Paraffin-embedded, formalin-fixed tissue
(biopsies, surgical samples)
2. Macro/microdissection can enhance sensitivity
Various test methods available, all adequate1. 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
KRAS Mutation Testing
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
2 3
4
Molecular conditions for anti-EGFR efficiency
at the receptor level
at the intracellular level
I. Activation of EGFR in the tumor: EGFR gain of copies, ligands overexpression
II. No downstream oncogenic activation:
KRAS (40%)
BRAF (10%)
PTEN (loss, 30%)
PIK3CA? (20%)
A sequential strategy:
KRAS
Cancer Res 2007
J Clin Oncol 2008
Cancer Res 2009
BRAF
PTEN
PIK3CA
downstream signaling
?
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
Perrone F 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)
110 metastatic colorectal cancers patients treated with the EGFR-targeted monoclonal
antibodies panitumumab or cetuximab
KRAS mutational status(evaluable n=109)
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**
*p<0.05
(p=0.019)
Mutated KRAS
32/109 (29%)
Wild-Type KRAS
77/109 (71%)
Responders 2* 20*
Non Responders 30* 57*
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
Frequency of mutations in PIK3CA and KRAS, and loss of PTEN protein expression
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)
Stage IV (predictive)
18q LOH
KRAS BRAF PIK3CA PTEN
experimental
clinical practice
GRAZIE
Silvio Veronese : SS Patologia Molecolare
H Niguarda Milano
Andrea Sartore Bianchi : Oncologia Falk
Salvatore Siena H Niguarda Milano
Alberto Bardelli : IRCC Candiolo