44th ASCO Annual Meeting May 30-June 3, 2008McCormick Place, Chicago, Illinois

Evaluation of PTEN expression in colorectal cancer (CRC) metastases (mets) and in

primary tumors as predictors of activity of cetuximab plus irinotecan

treatment

F. Loupakis1,6, L. Pollina2, I. Stasi1, G. Masi1, N. Funel2, M. Scartozzi3, I. Petrini4, D. Santini5, S. Cascinu3, A.

Falcone1,6.1Department of Oncology, Azienda USL 6 - Istituto Toscano Tumori Livorno,

Italy, 2Division of Pathology, AOUP, Pisa, Italy,

3Division of Medical Oncology, Azienda Ospedaliera Ospedali Riuniti,Università Politecnica delle Marche, Ancona, Italy,

4Division of Medical Oncology, AOUP, Pisa, Italy,5Division of Medical Oncology, Campus Biomedico University, Rome, Italy, 6Department of Oncology, Transplants and New Technologies in Medicine,

University of Pisa, Italy

Main EGFR signalling pathways

I have no relevant relationships to disclose

ASCO General COI

Fotios Loupakis, MD

PIP3

Ligands

RAS

RAF

MEK

ERK

Nucleus

PTEN

EGF receptor

PI3K

Main EGFR signalling pathways

Anti-EGFR MoABs

PIP2

AKTmTOR

PTEN

PIP3

Ligands

AKT

Nucleus

EGF receptor

mTOR

PIP2

• PTEN (phoshatase and tensin homologue deleted on chromosome 10) gene encodes a phosphatase, whose major substrate is PIP-3

• Loss of PTEN (mono or bi-allelic inactivation, but also epigenetic silencing) results in increased PIP-3 concentration

• Increase of PIP-3 leads to AKT hyperactivation

PROTECTION FROM APOPTOSIS

PTENPI3K

KRAS

Ligands

RAS

RAF

MEK

ERK

Nucleus

EGF receptor

• KRAS (human homolog of the Kirsten rat sarcoma-2 virus oncogene) encodes a small self-inactivating signal-transducing GTP-binding protein

• KRAS can harbor oncogenic mutations that yield a constitutively active protein

• Activated Ras stimulates Raf-1, which leads to MAPK phosphorylation

CELL PROLIFERATION

BACKGROUND BACKGROUND

Recent data suggest a possible predictive role for PTEN loss of expression, evaluated on primary tumors, in metastatic CRC patients treated with cetuximab-containing therapies

(Frattini M. et al. BJC 2007)

Several retrospective studies have underlined the role of KRAS mutations as predictors of resistance to EGFR MoAbs in CRC

(from Lievre A. et al. Can Res 2006 to Amado RG. et al. JCO 2008 and over…)

Recent data suggest a lack of correlation for EGFR, pAKT, MAPK tested with IHC on primary tumors and related metastases from CRC

(Scartozzi M. et al. JCO 2004 and Scartozzi M. et al. BJC 2007)

OBJECTIVESOBJECTIVES

To evaluate the correlation between primaries and related CRC mets in terms of PTEN immunoreactivity and KRAS mutational status

To investigate the role of PTEN loss and KRAS status in predicting the activity of cetuximab plus irinotecan, in terms of RR and PFS, for pre-treated metastatic CRC patients both on primary tumors and related mets

STUDY DESIGN and TREATMENTSTUDY DESIGN and TREATMENT

Cetuximab initial dose of 400 mg/sqm i.v. d1 followed by 250 mg/sqm i.v. weekly

or

500 mg/sqm i.v. d1 every 2 weeks.

Irinotecan 130-180 mg/sqm i.v. d1 every 2 weeks.

Retrospective evaluation of 102 EGFR-positive metastatic CRC patients treated with Cetuximab plus Irinotecan and progressed after previous Irinotecan-containing therapies

Centers: 4 Medical Oncology Divisions from Central Italy TREATMENT

DESIGN

METHODS: PTENMETHODS: PTEN

A

B

A: PTEN positive

B: PTEN negative

1 2 3

Intensity

1+ 2+ 3+

% + cells

0-25 25-50 >50

• IHC performed on 3 µm tissue section obtained from paraffin-embedded specimens

• anti-PTEN antibody clone 17.A, Neomarkers

• Scoring:

Score ≥4 PTEN positive

MATERIALSMATERIALS

102 patients 3 mm tissue sectionsfrom FFPE blocks of

primaries and/or mets

96Primary tumours

59Metastatic Sites

53Paired samples

PATIENTS’ CHARACTERISTICSPATIENTS’ CHARACTERISTICS

N %

Patients 102 -

Age, median (range) 62 (38-78) -

ECOG PS 0-1/2 90/12 88%/12%

Sex (M/F) 60/42 59%/41%

Previous CT (1/≥2 lines)

18/84 18%/82%

Multiple sites of disease

84 84%

Liver only mts 12 12%

DEFINITION of RESPONDERSDEFINITION of RESPONDERS

Responders: CR+ PR (RECIST CRITERIA) + SD6

SD6

patients clearly progressed on previous irinotecan-based regimen with a Time To Progression <3 months that obtained a SD (RECIST) lasting >6 months

ACTIVITYACTIVITY

N=102

n %

RC 1 1

RP 13 13

SD6 5 5

SD 38 37

PD 45 44

PTEN (on primaries): ACTIVITYPTEN (on primaries): ACTIVITY

N=85 (11 not evaluable)

PTEN + n=49

PTEN -n=36

Responders 11 (22%) 4 (11%)

NOT Responders

38 (78%) 32 (89%)

Responders vs NOT Responders

Fisher’s Exact Test: p=0.25

Concordance between primaries and mets: PTENConcordance between primaries and mets: PTEN

PTEN + Primary

PTEN - Primary

PTEN + Mets

16 (36%) 11 (24%)

PTEN - Mets

7 (16%) 11 (24%)

45 primaries and related mets evaluable

Concordance: 27/45 = 60% (95% CI: 46-74%)

Among a total of 155 IHC performed (96 primaries +

59 mets), the analysis has been repeated twice in 78

cases (50%), always confirming previous findings.

PTEN (on mets): ACTIVITYPTEN (on mets): ACTIVITY

N=55 (4 not evaluable)

PTEN + n=33

PTEN -n=22

Responders (8 RECIST + 5 SD6)

12 (36%) 1 (5%)

NOT Responders

21 (64%) 21 (95%)

Responders vs NOT Responders

Fisher’s Exact Test: p=0.008

Concordance between primaries and mets: Concordance between primaries and mets: KRASKRAS

KRAS wt Primary

KRAS mut Primary

KRAS wt Mets

24 (56%) 0 (0%)

KRAS mut Mets

2 (5%) 17 (39%)

43 primaries and related mets evaluable

Concordance: 41/43 = 95% (95% CI: 84-99%)

KRASKRAS (on primaries): ACTIVITY (on primaries): ACTIVITY

N=88 (8 not evaluable)

KRAS wt n=53

KRAS mutn=35

Responders (12 RECIST + 3 SD6)

13 (25%) 2 (6%)

NOT Responders

40 (75%) 33 (94%)

Responders vs NOT Responders

Fisher’s Exact Test: p=0.023

PTEN + (on mets) and PTEN + (on mets) and KRAS KRAS wt vs All Others: ACTIVITYwt vs All Others: ACTIVITY

N=45 (14 not evaluable)

PTEN + and KRAS wt

n=17

All Others

n=28

Responders (7 RECIST + 2 SD6)

8 (47%) 1 (4%)

NOT Responders

9 (53%) 27 (96%)

Responders vs NOT Responders

Fisher’s Exact Test: p=0.0008

PTEN (on mets) and PFSPTEN (on mets) and PFS

PTEN + Median PFS : 4.7 mos

PTEN - Median PFS : 3.3 mos

Logrank Test: p=0.005

HR=0.49 95% CI: 0.20-0.75

PTEN (on mets) and OSPTEN (on mets) and OS

PTEN + Median OS : 11.2 mos

PTEN - Median OS : 10.6 mos

Logrank Test: p=0.37

HR=0.76 95% CI 0.38-1.43

PTEN + (on mets) and PTEN + (on mets) and KRASKRAS wt vs All Others, PFS wt vs All Others, PFS

PTEN+ and KRAS wt Median PFS : 5.5 mos

All Others Median PFS : 3.8 mos

Logrank Test: p=0.001

HR = 0.42 95% CI: 0.17-0.65

PTEN + (on mets) and PTEN + (on mets) and KRASKRAS wt vs All Others, OS wt vs All Others, OS

PTEN+ and KRAS wt Median OS : 15.1 mos

All Others Median OS : 10.6 mos

Logrank Test: p=0.006

HR = 0.39 95% CI: 0.18-0.75

CONCLUSIONSCONCLUSIONS

Primaries and related mets from CRC differed in terms of PTEN immunoreactivity in 40% of cases.

KRAS mutations found on primaries are almost always (95% of cases) confirmed on mets. Such analysis may be ruled out on any available tumor sample.

Loss of PTEN tested on mets predicted lack of activity of cetuximab plus irinotecan combination treatment in metastatic CRC pts.

KRAS is confirmed to be a predictor of resistance to cetuximab plus irinotecan combination treatment in metastatic CRC pts.

The combination of PTEN IHC performed on mets and KRAS mutational analysis identified a subgroup of patients with higher chances of benefiting from cetuximab plus irinotecan treatment.

Thanks to all investigators and patients!Thanks to all investigators and patients!

LivornoLoupakis F, Stasi I, Cremolini C, Masi G, Cupini S, Baldi GG, Fornaro L, Falcone A.

Pisa Pollina L, Funel N, Petrini I, Ricci S, Campani D.

Ancona Pierantoni C, Scartozzi M, Cascinu S.

Milano Rulli E, Floriani I.

Urbino Ruzzo A, Magnani G.

Roma Schiavon G, Santini D, Tonini G.

fotiosloupakis@gmail.com