1. 10th World Congress on Gastrointestinal Cancer, June 25-28, 2008
CCIB, Barcelona, Spain
Loss of PTEN expression in colorectal cancer (CRC) metastases (mets) predicts lack of activity of cetuximab plus irinotecan treatment
F. Loupakis1,6, I. Stasi1, L. Pollina2, G. Masi1, G. G. Baldi1, L. Fornaro1,
G. Schiavon3, M. Scartozzi4, I. Petrini5, D. Santini3, N. Funel2, D. Campani2,
G. Tonini3, 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, Campus Biomedico University, Rome, Italy,
4Division of Medical Oncology, Azienda Ospedaliera Ospedali Riuniti,
Università Politecnica delle Marche, Ancona, Italy,
5Division of Medical Oncology, AOUP, Pisa, Italy,
6Department of Oncology, Transplants and New Technologies in Medicine, University of Pisa, Italy 1

2. Main EGFR signalling pathways
Ligands
Anti-EGFR MoABs
EGF receptor
RAS
PIP2
RAF
PI3K
PTEN
PIP3
MEK
ERK
AKT
mTOR
Nucleus

3. PROTECTION FROM APOPTOSIS
PTEN
Ligands
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
EGF receptor
PIP2
PI3K
PTEN
PIP3
AKT
Nucleus
mTOR

4. KRAS
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
Ligands
EGF receptor
RAS
RAF
MEK
ERK
Nucleus

5. 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 Van Cutsem E. et al. ASCO 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)

6. OBJECTIVES
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

7. STUDY DESIGN and TREATMENT
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
Cetuximab initial dose of 400 mg/sqm i.v. d1 followed by mg/sqm i.v. weekly or
500 mg/sqm i.v. d1 every 2 weeks.
Irinotecan mg/sqm i.v. d1 every 2 weeks.

8. METHODS: PTEN Score ≥4 PTEN positive
IHC performed on 3 µm tissue section obtained from paraffin-embedded specimens
anti-PTEN antibody clone 17.A, Neomarkers
Scoring: A B 1 2 3
Intensity 1+ 2+ 3+
% + cells
0-25
25-50
>50
A: PTEN positive
B: PTEN negative
Score ≥ PTEN positive

9. MATERIALS 102 patients 3 mm tissue sections from FFPE blocks of
primaries and/or mets 96
Primary tumours 59
Metastatic Sites 53
Paired samples

10. PATIENTS’ CHARACTERISTICS%
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%

11. DEFINITION 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

12. ACTIVITY
N=102 n % RC 1 RP 13
SD6 5 SD 38 37 PD 45 44

13. PTEN (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

14. Concordance between primaries and mets: PTEN
45 primaries and related mets evaluable
PTEN + Primary
PTEN - Primary
PTEN +
Mets
16 (36%)
11 (24%)
PTEN -
7 (16%)
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.

15. PTEN (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

16. Concordance between primaries and mets: KRAS
43 primaries and related mets evaluable
KRAS wt Primary
KRAS mut Primary
KRAS wt
Mets
24 (56%)
0 (0%)
KRAS mut
2 (5%)
17 (39%)
Concordance: 41/43 = 95% (95% CI: 84-99%)

17. KRAS (on primaries): ACTIVITY
N=88 (8 not evaluable)
KRAS wt
n=53
KRAS mut
n=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

18. PTEN + (on mets) and KRAS wt 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

19. PTEN (on mets) and PFS PTEN + Median PFS : 4.7 mos
Logrank Test: p=0.005
HR= % CI:

20. PTEN (on mets) and OS PTEN + Median OS : 11.2 mos
Logrank Test: p=0.37
HR= % CI

21. PTEN (on mets) among KRAS wt pts only, PFS
PTEN+ Median PFS : 5.3 mos
PTEN- Median PFS : 3.7 mos
Logrank Test: p=0.026
HR = % CI:

22. PTEN + (on mets) and KRAS 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 = % CI: 22

23. PTEN + (on mets) and KRAS 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 = % CI:

24. CONCLUSIONS
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.

25. Thanks to all investigators and patients!
Livorno
Loupakis 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.

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