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