1. USC/Norris Comprehensive Cancer Center
Mechanisms of Resistance to EGFR Inhibitors: Is It Time to Start Selecting Patients?
Heinz-Josef Lenz, MD
Professor of Medicine
and Preventive Medicine
Director, GI Oncology Program
USC/Norris Comprehensive Cancer Center
Eilat, January 10, 2008

2. What Patients Expect Today and More So In The Future
Drugs that work
Drugs that are safe
Doses that are right for them

3. How to Use Predictive or Prognostic Markers
When (Prognostic)
With what (Predictive)
How much (Predictive)
What combination (Predictive)

4. Overview of potential biomarkers predicting efficacy
EGFR
IHC detection
FISH detection
mutations
gene levels/polymorphisms
EGFR ligands (EGF, heregulin, epiregulin, amphiregulin)
K-ras
COX-2

5. EGF Receptor: A Rational Target for CRC Therapy
Ligand
Target for EGFR-cetuximab
Target for EGFT-TK inhibitor
EGFR-TK pY
GRB2 pY
P13K
SOS
RAS
RAF pY
STAT
MEK
PTEN
AKT
MAPK
The human epidermal growth factor receptor (HER-1/EGFR) signaling pathway is thought to play a key role in tumor growth and the progression of numerous cancers, including CRC. The HER-1/EGFR transmembrane proteins consist of an extracellular ligand-binding domain, an intracellular tyrosine-kinase (TK) domain, and a transmembrane region. Numerous ligands bind to the EGFR extracellular domain, which promotes receptor homo- or heterodimerization with another EGFR receptor or other HER family members. This induces activation of the receptor’s TK activity and initiates a downstream signaling cascade that ultimately leads to tumor cell proliferation, migration, adhesion and angiogenesis, and cell death inhibition (apoptosis).
In CRC, HER-1/EGFR signaling is dysregulated. HER-1/EGFR overexpression has also been correlated with disease progression, poor prognosis, and reduced sensitivity to chemotherapy. Therefore, several agents have been developed to target HER-1/EGFR, including small-molecule TK inhibitors and monoclonal antibodies (mAbs). Currently, cetuximab is the most advanced mAb in develpoment for CRC.
References
Meyerhardt JA, Mayer RJ. Systemic therapy for colorectal cancer. N Engl J Med. 2005;352: ; Venook A. Critical evaluation of current treatments in metastatic colorectal cancer. Oncologist. 2005;10:
Gene transcription
Cell-cycle progression P P
Cyclin D1
MYC
JUN
FOS
Proliferation/ maturation
MYC
Cyclin D1
Survival (anti-apoptosis)
Chemotherapy/ radiotherapy resistance
Invasion and
metastasis
Angiogenesis
Meyerhardt JA, Mayer RJ. N Engl J Med. 2005;352: ; Venook A. Oncologist. 2005;10:

6. Objective Response N (%)
Reference
Treatment (panitumumab or cetuximab)
No of patients (WT:MT) MT WT
A. Liévre, et al. (AACR Proceedings, 2007)
cmab ± CT
76 (49:27)
0 (0)
24 (49)
S. Benvenuti, et al. (Cancer Res, 2007)
pmab or cmab or cmab + CT
48 (32:16)
1 (6)
10 (31)
W. De Roock, et al. (ASCO Proceedings, 2007)
cmab or cmab + irinotecan
113 (67:46)
27 (40)
D. Finocchiaro, et al. (ASCO Proceedings, 2007)
81 (49:32)
2 (6)
13 (26)
F. Di Fiore, et al. (Br J Cancer, 2007)
cmab + CT
59 (43:16)
12 (28)
S. Khambata-Ford, et al. (J Clin Oncol, 2007)
cmab
80 (50:30)
5 (10)

7. Objective Tumor Response (Central Radiology)
KRAS
All Evaluable
n (%)
Mutant
Wild-type
Response
Pmab
(N = 208)
BSC
(N = 219)
(N = 84)
(N = 100)
(N = 124)
(N = 119) CR
0 (0) PR
21 (10)
21 (17) SD
52 (25)
22 (10)
10 (12)
8 (8)
42 (34)
14 (12) PD
104 (50)
149 (68)
59 (70)
60 (60)
45 (36)
89 (75)
CR, PR, SD
73 (35)
63 (51)
Pmab, panitumumab; BSC, best supportive care; CR, complete response; PR partial response; SD, stable disease; PD, disease progression

8. Primary diagnosis, n (%) Colon cancer 286 (67) 118 (64) 168 (69)
KRAS Evaluable
Mutant KRAS
Wild-type KRAS
Total (N = 427)
Total (N = 184)
Total (N = 243)
Sex, n (%)
Men
270 (63)
111 (60)
159 (65)
Baseline age, years
Median (min, max)
62.0 (27, 83)
63.0 (29, 82)
Primary diagnosis, n (%)
Colon cancer
286 (67)
118 (64)
168 (69)
Months since primary diagnosis
Median
25.1
24.1
ECOG performance status, n (%)
0-1
366 (86)
155 (84)
211 (87)
≥ 2
61 (14)
29 (16)
32 (13)
Prior adjuvant chemotherapy, n (%)
Yes
149 (35)
67 (36)
82 (34)
Cells with EGFr membrane staining, n (%)
1% to < 10%
103 (24)
43 (23)
60 (25)
10% to 100%
322 (75)
140 (76)
182 (75)
ECOG, Eastern Cooperative Oncology Group; EGFr, epidermal growth factor receptor

9. Distribution of KRAS Mutations
Panitumumab + BSC
BSC alone
Total
Mutation n %
12 Ala 8
9.5 7
7.0 15
8.2
12 Asp 34
40.5 36
36.0 70
38.0
12 Arg
0.0 3
3.0
1.6
12 Val
17.9 25
25.0 40
21.7
12 Cys
8.3 14
7.6
12 Ser 5
6.0 9
9.0
13 Asp
14.0 29
15.8

10. K-RAS mutation status trends with Progression Free Survival
Patients Free of Tumor Progression (%)
Wildtype
Mutant
(days)
Hazard ratio =
95% CI = to
Ford et al AACR 2007

11. Mutant KRAS Subgroup: PFS by Treatment
Median
In Weeks
Mean
In Weeks
1.0
Events/N (%)
0.9
Pmab + BSC
76/84 (90)
7.4
9.9
0.8
BSC Alone
95/100 (95)
7.3
10.2
0.7
HR = 0.99 (95% CI: 0.73–1.36)
0.6
Proportion with PFS
0.5
0.4
0.3
0.2
0.1
0.0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52
Patients at Risk
Weeks
Pmab + BSC 84 78 76 72 26 10 8 6 5 5 5 5 4 4 4 4 2 2 2 2 2 2 2 1 1 1
BSC Alone
100 91 77 61 37 22 19 10 9 8 6 5 5 4 4 4 4 4 4 3 3 3 2 2 2 2

12. Wild-type KRAS Subgroup: PFS by Treatment
p < for quantitative-interaction test comparing PFS log-HR (pmab/BSC) between KRAS groups . 1 2 3 4 5 6 7 8 9 10 12 14 16 18
Weeks
124
115/124 (93)
12.3
19.0
114/119 (96)
7.3
9.3
Pmab + BSC
BSC Alone
Events/N (%)
Median
In Weeks
Mean
HR = 0.45 (95% CI: 0.34–0.59)
Stratified log-rank test, p <
Patients at Risk
119
112
106 80 69 63 58 50 45 44 33 25 21 20 17 13
109 91 81 38 15 11
Proportion with PFS

13. Overall Survival by Treatment and KRAS
Median
In Months
Events/N (%)
1.0
Pmab–Wild-type
107 / 124 (86)
8.1
0.9
BSC–Wild-type
110 / 119 (92)
7.6
0.8
Pmab–Mutant
79 / 84 (94)
4.9
0.7
BSC–Mutant
95 / 100 (95)
4.4
0.6
Survival Probability
0.5
0.4
HR = 0.67 (95% CI: 0.55–0.82)
(adjusted for treatment and
randomization factors; ECOG, region)
0.3
0.2
0.1
0.0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34
Months

14. Why more effective in refractory patients?
First Line
FOLFORI
Second Line
CPT-11
Third Line
Benefit 8%
12%
23%
No Cetuximab
36% 4% NA
Cetuximab
44%
16%

15. Is it Patient or Tumor Selection?
Patient Selection
Patients who are being able to receive more than three lines of therapy are overall doing better (less ras mutations?)
Tumor Selection
Tumor biology changes with increasing exposure to chemotherapy
Exposure to specific cytotoxic changes EGFR status
EGFR expression associated with Metastatic Potential and Poor Prognosis
Addiction to EGFR with more tumor progression

16. Prediction or Prognosis?

17. Rak, J. et al. Cancer Res 2000;60:490-498
Copyright ©2000 American Association for Cancer Research

18. Kras mutations don’t come alone!
Kras mutations associated with braf mutations which itself with CIMP
Kras mutations associated with PI3K mutations

19. EGFR mutations
Biopsy samples from 110 patients with mCRC enrolled in a ERBITUX monotherapy trial revealed no mutations in EGFR (exons 18–21)
Khambata-Ford S, et al. J Clin Oncol 2007;25:3230–3237

20. EGFR inhibitors: EGFR expression (FISH)
EGFR expression determined by FISH may have predictive value for ERBITUX therapy1,2 and panitumumab therapy 2,3
BUT – no association with response was noted in another recent study4
1.0
1.0
p=0.05
EGFR FISH+
EGFR FISH-
TTP (months)
Cumulative distribution function
Cumulative survival function
p=0.7
Survival time (months)
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
1. Cappuzzo F, et al. Ann Oncol 2007 (Epub ahead of print); 2. Moroni M, et al. Lancet 2005;6:279–286; 3. Sartore-Bianchi A, et al. J Clin Oncol 2007;25:3238–3245; 4. Personeni N, et al. J Clin Oncol 2007;25:18S (Abstract No )

21. EGFR gene levels: Effect on survival
95% CI: 4.4–13.5 months 8
7.3 months
95% CI: 1.7–4.5 months 6
Median survival (months) 4
2.2 months 2
p=0.09
Low
High
mRNA levels
There was no significant correlation between EGFR gene expression level and response to ERBITUX (small sample size)
39 patients with mCRC refractory to irinotecan and oxaliplatin treated with ERBITUX monotherapy
Vallböhmer D, et al. J Clin Oncol 2005;23:3536–3544

22. Progression-free survival (months)
EGFR gene copy number and efficacy of Cetuximab
There was no relationship between EGFR gene copy number and either response or PFS
 Ct values were related to survival using  Ct as a fixed covariate in a Cox model of survival (Wald 2 P=0.03)
3.0
2.0
1.0
0.0
-1.0
-2.0
3.0
2.0
1.0
0.0
-1.0
-2.0
 Ct value (dCt)
 Ct value (dCt)
PR SD PD
Progression-free survival (months)
Best response
Ct = threshold cycle; = censored patients
Lenz, H.-J. et al. J Clin Oncol 2006;24:4914–4921

23. Mechanisms of gene amplification
Double minutes
Amplified chromosome regions
Distributed across genome
Albertson DG. Trends Genet 2006;22:447–453

24. EGFR ligand expression: a predictor for increased PFS?
140
P=0.0002
EREG
120
days
AREG
100
days 80
Median PFS (days)
P=0.0002 60
57 days
57 days 40 20
High
Low
EGFR ligand expression
A similar, significant relationship was also noted between ligand expression and disease control
n=110, ERBITUX monotherapy
Khambata-Ford S, et al. J Clin Oncol 2007;25:

25. EGFR pathway gene polymorphisms
EGFR codon R497K
EGFR CA dinucletide repeat in intron 1
EGF A61G
COX-2 G-765C
Interleukine-8(IL-8) T-251A
VEGF C936T
Cyclin D1 A870G

26. Germline Polymorphisms
Translation
Splicing
exon 1
exon 2
3’UTR
5’UTR
promoter
Transcription
SNP in untranslated region
or promoter region
SNP in coding region of exon
RNA stability
SNP at splice site 26

27. Response to Cetuximab COX2 PolymorphismsPR SD PD 0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
G/G
G/C
C/C
(n=78)
(n=30)
(n=4)
Response to Cetuximab by COX GC polymorphism P
value = 0.097

28. COX2 Polymorphisms associated with PFS in mCRC treated with cetuximab
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0 3 6 9 12
Months Since Start of Cetuximab Treatment
Estimated Probability of Progression-Free Survival
Log-rank P
value = 0.031
COX2 765GC Polymorphism and PFS
G/G (n=87)
G/G (n=34)
C/C (n=4)

31. Antibody-dependent cell-mediated cytotoxicity (ADCC)
Courtesy of Dr. Arteaga

32. Polymorphisms of FCGamma 2a and 3a associated with PFS
Zhang et al JCO 2007

33. Fc Gamma Receptor 3a polymorphism associated with response to Cetuximab and Bevacizumab (BOND 2)
Lenz et al ASCO GI 2007 abstract 401

34. Mechanisms of Resistance
Kras a marker of resistance, but also prognostic marker?
Kras patient/tumor selection? Associated with PI3K mutations?
Horizontal and vertical inhibition to further increase efficacy and overcome resistance
Cox2 a promising target for combination with EGFR inhibitors
ADCC a mechanism of efficacy of IgG1 AB

35. Collaborations Medical Oncology: Syma Iqbal, Anthony El-Khoueiry
Surgery: Robert Beart, Richard Selby
Danenberg Lab: Peter Danenberg
ResponseGenetics: Kathy Danenberg
Lenz Lab: Philipp Manegold Zhang Wu Anne Schultheiss Mizutomo Azuma Georg Lurje Alexandra Pohl
Fumio Nagashima
Mol Epidemiol: Chris Haiman, Robert Haile
Statistics: Susan Groshen, Dongyun Yang
Pathology: Robert Ladner, William Fazzone, Peter Wilson, Melissa LaBonte
MSKCC: Len Saltz, M Shah