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Molecular Predictors in Clinical Decision Making for Colon
Cancer
Axel Grothey
Professor of Oncology
Mayo Clinic Rochester
Disclosures
• Consulting activities (honoraria went to the Mayo Foundation)• Amgen• Bayer• Pfizer• Roche/Genentech• Sanofi-Aventis• BMS• Eli Lilly/Imclone
I WILL include discussion of investigational or off-label use of a product in my presentation.
Landis SH, et al. CA Cancer J Clin 1999;49:8–31
CRC: worldwide incidence• Third leading cancer death in the world• Second in the US and Western Europe
CRC = colorectal cancer
Sporadic
Lynch Syndrome
Familial
Hereditary
FAP; AFAPMixed Polyposis SyndromeAshkenazi I1307KCHEK2 (HBCC)MYHTGFBR1
PJSFJPCDBRRS
= as yet undiscovered hereditary cancer variants
HamartomatousPolyposis
Syndromes
AC-1 without MMR(Familial CRC of syndrome “X”)
Genetic Syndromes: Magnitude of the Problem
Annual worldwide incidence of CRC is 1,023,152*:
• Lynch syndrome (LS) accounts for 2-5% (20,460-51,160 cases).
• < 1% (10,230 cases) constitute FAP.
• 20% (204,630 cases) are familial (2 or more first- degree relatives with CRC.
*International Agency for Research on Cancer. Globocan 2002. Available at: http://www-dep.iarc.fr/.
H. Lynch, ASCO 2009
Genetic Heterogeneity in HNPCC
HNPCC is associated with germline mutations in any one of at least five genes
Chr 2Chr 3
Chr 7
MSH2
PMS1
MLH1PMS2
MSH6
Normal epithelium
Early adenoma
Intermed. adenoma
Late adenoma
Carcinoma
Hypo-methylation
Hyper-methylation
Late adenoma
CarcinomaMLH1MSH 2/3/6
18q lossSMAD2/4 TP53
KRASAPCb-catenin
WNT-pathway
MAPK-pathway
TGFb-pathway
P53-BAX-pathway
TGFIIbBAXTCF4WISP3
P53-BAX-WNT-TGFb-
pathway
15% microsatellite instable tumors (MSI)
85% chromosome instable tumors (CIN)
SPORADIC CRC
12.3% of patients presented with recurrent CRC.*2002 data.
24.5% stage II*
18.6% stage IV*12% stage I*
32.6% stage III*
American Cancer Society, 2005; Datamonitor, 2003.
CRC:Demographics and Presentation
• Estimated 2009 U.S. incidence (new cases): 147,000
• Estimated 2008 U.S. mortality: 49,900
Palliative Therapyof Colorectal Cancer
Advances in the Treatment of Stage IV CRC
1980 1985 1990 1995 2000 2005
5-FUIrinotecan
CapecitabineOxaliplatin
CetuximabBevacizumab
Best supportive care (BSC)
median overall survival
Panitumumab
Treatment paradigms for mCRC
• Some patients with stage IV disease can be cured by an interdisciplinary approach
• In the palliative setting: FOLFOX = XELOX = FOLFIRI (XELIRI has problems with toxicity)
• Most patients tolerate a chemotherapy doublet, but not all need it
• The addition of biologics to chemotherapy has improved outcomes, but not as much as we hoped
• We are on the verge of individualized therapy based on molecular predictive factors
Personalized Cancer Therapy: Key Challenges
• Tumor heterogeneity• Primary vs metastasis• Established tumor vs micrometastasis
• Whack-a-mole pathway modulation
• Combination therapy complicates choice of appropriate biomarkers
• Identification of biomarkers lags behind standard of care and agents used in clinical trials
• Most biomarkers identified in retrospective analysis without prospective validation
• Complex, step-wise trial designs to validate usefulness of biomarkers
Prognostic markerRetrospective
analysis
Predictive markerRandomized Clinical Trials
Biomarker Validation
5-FU: Predictive Markers
FUH2
FUPA
FBAL
DPDFUrd FUMP FUDP FUTP
FUdR
FdUMP FdUDP FdUTP
dUMP dTMP
5,10-CH3THF DHF
DNA
RNA
FU
TS
LV
TP
DPD, TS and TP Gene Expression vsResponse to 5-FU/LV in Colorectal Cancer
0
0.2
0.4
0.6
0.8
1
1.2
13
5
13
7
15
0
15
4
16
5
20
4
28
9
36
1
37
4
57
4
43
8 7
91
12
1
15
2
16
4
18
9
19
6
21
7
22
0
27
0
27
8
28
8
35
9
39
6
40
1
45
8
52
6
55
9
58
2
58
3
58
5
10
5m
DPD
TS
TP
Response Non response
Patient ID Number
Danenberg
Tum
or
Pro
file
Sca
le
Salonga et al. Clin Cancer Res 2000
Single marker analysis - Chemotherapy
Agent Marker PrognosticPredictive
Efficacy Toxicity
5-FU TS + +
DPD (+) +
TP (+)
Irinocetan UGT1A1 +
Oxaliplatin GSTP1 + +
ERCC1 + +
XPD (ERCC2) +
Murine Ab“momab”
ChimericMouse-Human Ab
“ximab”
Humanized Ab“zumab”
Fc
Fab
Human Ab“mumab”
Biologic Agents in Colorectal Cancer = Monoclonal Antibodies
(17-1A) Cetuximab Bevacizumab
PanitumumabEGFR
VEGF
Nomenclature of Monoclonal Antibodies
-mab monoclonal antibody
-mo-mab mouse mab
-xi-mab chimeric mab
-zu-mab humanized mab
-mu-mab human mab
-tu-xx-mab tumor-directed xx mab
-li-xx-mab immune-directed xx mab
-ci-xx-mab cardiovascular-directed xx mab
-vi-xx-mab virus-directed xx mab
Inf-li-xi-mab Beva-ci-zu-mab Ri-tu-xi-mab Pani-tu-mu-mab
mAbs Target Tumor Cell-Bound EGFR
Extracellular
Intracellular
Ligand
EGF-R
PI3K
Akt
Raf
MEK
MAPK
Cell Motility
MetastasisAngiogenesisProliferation
Cell survivalDNA
PTEN
Ras
mAbs Target Tumor Cell-Bound EGFR
Extracellular
Intracellular
Ligand
EGF-R
PI3K
Akt
Raf
MEK
MAPK
Cell Motility
MetastasisAngiogenesisProliferation
Cell survivalDNA
Ras
PTEN
RAS (RAt Sarcoma virus)
• Three genes encode highly homologous proteins: H-RAS, N-RAS, and K-RAS
• Point mutations in RAS genes occur in 30% of all cancers
• K-RAS mutations present in 40% of CRC• Codons 12, 13, and 61 are most commonly
involved• Mutations result in constitutive activation of
RAS-RAF-MAPK signaling pathway leading to cell proliferation and enhanced cell survival
KRAS as Biomarker for Panitumumab Response in Metastatic CRC
• PFS log HR significantly different depending on K-ras status (P < .0001)• Percentage decrease in target lesion greater in patients with wild-type KRAS
receiving panitumumab
Patients With Mutant KRAS
Meanin Wks
Stratified log rank test: P < .0001
115/124 (93)
Patients With Wild-Type KRAS
1.0
0.9
Pro
po
rtio
n W
ith
PF
S
0.8
0.70.60.50.4
0.3
0.20.1
00 2 4 6 8 10
Events/N (%)Medianin Wks
Pmab + BSCBSC alone
114/119 (96)
12.37.3
19.09.3
HR: 0.45 (95% CI: 0.34-0.59)
12 14 16 18 20 22 24 26 2830 32 3436 38 4042 44 46 48 50 52
Weeks
Pro
po
rtio
n W
ith
PF
S
1.0
0.90.8
0.70.60.50.4
0.30.20.1
00 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 3436 38 4042 44 46 48 50
Weeks
Pmab + BSCBSC alone Mean
in Wks
76/84 (90)
Events/N (%)Medianin Wks
95/100 (95)
7.47.3
9.910.2
HR: 0.99 (95% CI: 0.73-1.36)
52
Amado et al. JCO 2008
NCIC CTG CO.17: Randomized Phase III Trial in mCRCCetuximab vs BSC (no cross-over)
KRAS mut KRAS wild-type All patients
BSC
n=83
Cetux
n=81
BSC
n=113
Cetux
n=117
BSC
n=285
Cetux
n=287
RR 0% 1.2% 0% 12.8% 0% 6.6%
PFS (mos) 1.8 1.8 1.9 3.8 1.8 1.9
OS (mos) 4.6 4.5 4.8 9.5 4.6 6.1
Karapetis et al. NEJM 2008
<0.0001
<0.0001 <0.0001
0.0046
NCIC CTG C0.17: Overall Survival by KRAS Status in BSC Arm
HR 1.01 95% CI (0.74,1.37)
Log rank p-value: 0.97
KRAS status MS (months)
95% CI
Mutated 4.6 3.6 – 5.5
Wild-Type 4.8 4.2 – 5.5
Karapetis et al. NEJM 2008
CRYSTAL Study (1st Line)
FOLFIRI + Cetuximab
FOLFIRI
EGFR-expressingmetastatic CRC PFS
Stratified by:• Regions • ECOG PS
• Primary Endpoint: PFS (independent review)
• Secondary Endpoints: RR, DCR, OS, Safety, QoL
• Sample Size: 1217 patients randomized, ITT: 1198 pts
N = 599
N = 599
Van Cutsem et al. NEJM 2009
R
CRYSTAL trial: Primary endpoint PFS ITT population independent review
Progression-free survival time (months)
PF
S e
stim
ate
1.0
0.8
0.9
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 2 4 6 8 10 12 14 16 18 20
8.9 mo
8.0 mo
FOLFIRI
FOLFIRI + Cetuximab
1-year PFS rate23% vs 34%
Subjects at risk
FOLFIRI alone 599 492 402 293 178 83 35 16 7 4 1Cetuximab + FOLFIRI
599 499 392 298 196 103 58 29 12 5 1Van Cutsem et al. NEJM 2009
HR = 0.851P = 0.0479
RR
46.9%
38.7%
P = 0.0038
Relating KRAS status to efficacyPrimary endpoint: PFS – KRAS wild-type
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0 2 4 6 8 10 12 14 16 18
Months
Pro
gre
ss
ion
-fre
e s
urv
iva
l e
sti
ma
te
Cetuximab + FOLFIRI FOLFIRI
KRAS wild-type (n=348) HR=0.68; p=0.017
mPFS Cetuximab + FOLFIRI: 9.9 months mPFS FOLFIRI: 8.7 months
1-yr PFS rate25% vs 43%
Van Cutsem et al. NEJM 2009
CRYSTAL: recent efficacy updateafter additional KRAS testing
Van Cutsem et al. ESMO-ECCO 2009, ASCO GI 2010
KRAS wild-type FOLFIRI FOLFIRI + cetuximab
P value
n 350 316
RR (%) 39.7 57.3 < 0.0001
mPFS (mos) 8.4 9.9 0.0012
mOS (mos) 20 23.5 0.0094
KRAS mutated FOLFIRI FOLFIRI + cetuximab
P value
n 183 214
RR (%) 36.1 31.3 0.34
mPFS (mos) 7.7 7.4 0.26
mOS (mos) 16.7 16.2 0.75
Courtesy: C. Punt
Δ 1.5Δ 3.5
OPUS: Study design
Primary endpoint
• Overall confirmed response rate (as assessed by independent review)
Secondary endpoints
• PFS time
• OS time
• Rate of curative surgery for metastases
• Safety
Cetuximab + FOLFOX4
N=168
FOLFOX4
N=169
EGFR-detectablemCRC
Stratification by:
ECOG PS 0/1, 2
R
Bokemeyer et al. JCO 2008
OPUS - Relating KRAS status to efficacySecondary endpoint: PFS – KRAS wild-type
0.5
1.0
0.4
0.3
0.2
0.1
0.0
0.6
0.7
0.8
0.9
80 2 4 6 10 12
Months
mPFS Cetuximab + FOLFOX: 7.7 mosFOLFOX: 7.2 mosHR=0.57; p=0.016
FOLFOX
Cetuximab + FOLFOXPro
gre
ssio
n-f
ree
surv
ival
es
tim
ate
Bokemeyer et al. JCO 2008
OPUS - Relating KRAS status to efficacySecondary endpoint: PFS – KRAS mutant
0.5
1.0
0.4
0.3
0.2
0.1
0.0
0.6
0.7
0.8
0.9
80 2 4 6 10 12
Months
Pro
gre
ssio
n-f
ree
surv
ival
es
tim
ate
mPFS Cetuximab + FOLFOX: 5.5 mosFOLFOX: 8.6 mosHR=1.83; p=0.019
FOLFOX
Cetuximab + FOLFOX
Bokemeyer et al. JCO 2008
PRIME: FOLFOX +/- P-mab PFS by KRAS Mutation Status
WT KRAS MT KRAS
Eventsn (%)
Median (95% CI) months
Panitumumab + FOLFOX
199 (61) 9.6 (9.2–11.1)
FOLFOX 215 (65) 8.0 (7.5–9.3)
HR = 0.80 (95% CI: 0.66–0.97) P-value = 0.02
Eventsn (%)
Median (95% CI) months
Panitumumab + FOLFOX
167 (76) 7.3 (6.3 – 8.0)
FOLFOX 157 (72) 8.8 (7.7 – 9.4)
HR = 1.29 (95% CI: 1.04 – 1.62)P-value = 0.02
Months
Prop
ortio
n Ev
ent-F
ree
0
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
MonthsPr
opor
tion
Even
t-Fre
e
0
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
Douillard, et al. ECCO-ESMO 2009
COIN (cetuximab): First-line Study
Continuous* XELOX or FOLFOX Arm A
RFirst-line mCRC
(n= 2445)Arm B
Continuous XELOX
or FOLFOX + cetuximab
Arm CIntermittent**XELOX or FOLFOX
*Treatment until disease progression or unacceptable toxicity**Stop and Go treatment (12 wks then restart at progression)
MRC-sponsored study supported by Merck (109 UK/Irish Hospitals)
65% XELOX; 35% FOLFOX(patient/physician choice)
• Primary endpoints: • OS in patients with K-ras wild-type tumours
• Secondary endpoints include:• OS in K-ras mutant; “all” wild-type
(K-ras, N-ras, B-raf); “any” mutant, ITT• PFS• Response rate• Quality of life• Health economic evaluation
Maughan, et al. ECCO-ESMO 2009
1.00
0.75
0.50
0.25
0
Survival probability
Time (months)
COIN study: KRAS WT PFS
No. at riskArm A Arm B
0 6 12 18 24 30 36 42
367361
245249
92103
4142
1822
119
66
10
Arm A (XELOX/FOLFOX)Arm B (XELOX/FOLFOX + cetuximab)
Arm A Arm B Diff.
Median PFS, months
8.6 8.6 +0.07
HR point estimate = 0.95995% CI 0.84–1.09
p=0.60
Maughan, et al. ECCO-ESMO 2009
Rationale for Combining EGFR- and Angiogenesis- Inhibitors
EGFR Inhibitors• Tumor cell growth • Synthesis of angiogenic
proteins
• Response of endothelial cells to angiogenic proteins
Tumor
Angiogenesis Inhibitors
Angiogenic proteinsbFGFVEGFTGF-
Endothelial cells
Herbst et al. J Clin Oncol. 2005;23:2544.
- - -
Targets
CAIRO2: Study design
Primary endpoint
• Progression-free survival
Secondary endpoints
• RR
• OS time
• Toxicity
• Translational research
CapOx + BEV
(COB, n=368)
CapOx + BEV + Cetuximab
(COB-C, n=368)
EGFR-detectablemCRC R
Tol et al. NEJM 2009
Oxaliplatin d/c’d after 6 cyclesi.e. after 18 weeks = 4.5 mos
COBn = 368
COB-Cn = 368
P-value
Median PFS (mos)(HR; 95% CI)
10.7 (9.7-12.3)
9.4 (8.4-10.5)
0.01
Median OS (mos)(HR; 95% CI)
20.3 (17.8-24.7)
19.4(17.5-21.4)
0.16
Response rate(CR + PR)
50% 52.7% 0.49
Disease control rate(CR + PR + SD)
94% 94.6% 0.72
CAIRO2 – Summary Efficacy results
Tol et al. NEJM 2009
KRAS wild-typen = 314 (61%)
KRAS mutatedn = 196 (39%)
p value
Median PFS (months)
COB 10.6 12.5 0.80
COB-C 10.5 8.1 0.04
p value 0.30 0.003
Median OS (months)
COB 22.4 24.9 0.82
COB-C 21.8 17.2 0.06
p value 0.64 0.03
CAIRO2 - KRAS genotyping (n=501)
Tol et al. NEJM 2009
mAbs Target Tumor Cell-Bound EGFR
Extracellular
Intracellular
Ligand
EGF-R
PI3K
Akt
Raf
MEK
MAPK
Cell Motility
MetastasisAngiogenesisProliferation
Cell survivalDNA
Ras
PTEN
Distribution of Mutations in COIN
Maughan, et al. ECCO-ESMO 2009
Advances Towards Personalized Therapy:What about other Molecular Biomarkers?
• EGFR gene copy number (FISH)
• EGFR ligand overexpression – epiregulin and amphiregulin
• BRAF mutations
• PI3K/Akt mutations
• PTEN expression
• Expression of alternative, vertical signaling pathways - e.g. IGF-1R
Fluorescence in-situ hybridization (FISH) for detecting EGFR expression
• FISH analysis can show increases in EGFR copy number
• Significantly increased copy number may be associated with clinical response
• One study demonstrated
– 8/9 responders had increased EGFR copy number vs 1/21 non-responders
• Costly and technically challenging testing method
• Further studies required
Moroni et al, Lancet Oncol 2005
Epiregulin and Amphiregulin Levels Correlate With Disease Control
Subjects
CR/PR SD Non-Responders
Khambata-Ford S et al, JCO 2007
0
1000
2000
3000
4000
5000
6000
7000
Affy
me
trix
mR
NA
leve
l
0
500
1000
1500
2000
2500
3000
3500
Affy
me
trix
mR
NA
leve
l
Amphiregulin
Epiregulinp = 1.5e-05
p = 2.5e-05
Epiregulin/Amphiregulin Levels Correlate With PFS in mCRC
Pa
tien
ts F
ree
of T
um
or
Pro
gre
ssio
n (
%)
100
80
60
40
20
0
0 100 200 300 400
Time (days)
Hazard ratio = 0.455895% CI = 0.2284 to 0.6177
EREGp = 0.0001
EREG expression cutoff 500 High Low
Pa
tien
ts F
ree
of T
um
or
Pro
gre
ssio
n (
%)
100
80
60
40
20
0
0 100 200 300 400
Time (days)
Hazard ratio = 0.467995% CI = 0.2204 to 0.6247
AREG expression cutoff 100 High Low
AREGp = 0.0002
Khambata-Ford S et al, JCO 2007
Role of PI3K Pathway• 40% of CRC tumors have
mutations in PI3K pathway1
• PI3K pathway dysregulation predicts Cetuximab resistance in CRC cell lines2
• Of 36 tumors with PI3KCA mutations, 27 also had alteration in KRAS1
• Patients treated with Cetuximab3
• 4/31 PI3KCA mutations (4/16 non-responders)
• 4/31 PTEN gene copy number
• 3/30 PTEN mutations (3/15 non-responders)
• PI3KCA mut: early or late event?
1. Parsons, et al. Nature 2005. 2. Jhawer, et al. Cancer Res 2008; 3. Perrone, et al. Ann Oncol 2009
RTKs
P P PP P P PP P PP
P
P
IRS2 p85PIK3CA
PTEN
PDK1 AKT2
PAK4
PIP3PIP2 PIP3
?
Tumours PKK1 AKT2 PAK4AKT2/ PAK4
amp IRS2 amp INSRR ERBB4 PTEN PIK3CA
CSX3 T354M wt wt wt wt wt wt wt wt
CX10 T354M wt wt wt wt wt wt wt wt
MX20 D527E wt wt wt wt wt wt wt wt
CX7 wt S302G wt wt wt wt wt wt wt
HX66 wt R371H wt wt wt wt wt wt wt
CO86 wt wt A279T wt wt wt wt 800del/968del wt
HX63 wt wt E329K wt wt wt wt wt wt
CO78 wt wt wt 15 fold wt wt wt wt wt
CO82 wt wt wt 8 fold wt wt wt wt wt
CO84 wt wt wt wt 12 fold wt wt wt wt
CO69 wt wt wt wt 7 fold wt wt wt wt
HX160 wt wt wt wt 6 fold wt wt wt wt
MX5 wt wt wt wt wt T1014M wt wt wt
CO87 wt wt wt wt wt wt I1030M wt wt
MX9 wt wt wt wt wt wt wt 904-919del wt
CX28 wt wt wt wt wt wt wt Y88C wt
HX170 wt wt wt wt wt wt wt L325H/LOH wt
HX199 wt wt wt wt wt wt wt R741/F341V R88Q
HX219 wt wt wt wt wt wt wt A86P/LOH wt
HX242 wt wt wt wt wt wt wt R47S wt
36 cases wt wt wt wt wt wt wt wt MUT
90 cases wt wt wt wt wt wt wt wt wt
Mutations of PI3K pathway genes in CRC
PIK3CA Point Mutations
Bader et al., Nat Rev Cancer 2005
Hotspots
Exon 9
Exon 20
CRC
Sartore-Bianchi A et al, Cancer Res 2009
PFS and PIK3CA Mutational Status in mCRC Patients Treated With Panitumumab/Cetuximab
Cetuximab 13%
Panitumumab 20%
Cetuximab/Irinotecan 67%
110 pts> 85% received at least 1 prior
Rx
Prenen H et al, Clin Cancer Res 2009
Fig. 1
Cetuximab 16
Cetuximab/Irinotecan 184
Total Patients 200
Cape +/- Perifosine Rand Phase II
Cycle = 21 Days
Primary Objective:
– To compare time to progression (TTP) of P-CAP vs. CAP as 2nd or 3rd line Rx
Secondary Objective:
– To compare overall response rate (CR + PR) and overall survival
– To evaluate the safety of P-CAP vs. CAP
Patients with 2nd or 3rd line mCRC
No prior Rx with CAP in metastatic setting
Prior Rx with 5-FU or 5-FU based regimen
Richards et al., ASCO 2010
Median Time to Progression (TTP)5-FU REFRACTORY
PATIENTS
Median TTP: P-CAP:18 weeks [95% CI (12, 36)]
Median TTP: CAP: 10 weeks [95% CI (6.6, 11)]
p-value = 0.0004
Hazard ratio: 0.186(0.066, 0.521)
ALL EVALUABLE PATIENTS
Median TTP: P-CAP: 28 weeks [95% CI (12, 48)]
Median TTP: CAP: 11 weeks [95% CI (9, 15.9)]
p-value = 0.0012
Hazard ratio: 0.284(0.127, 0.636)
Richards et al., ASCO 2010
Median Overall Survival (OS)5-FU REFRACTORY
PATIENTS
Median OS: P-CAP:15.1 mos [95% CI (7.3, 22.3)]
Median OS: CAP: 6.6 mos [95% CI (4.7, 11.7)]
p-value = 0.0112
Hazard ratio: 0.313(0.122, 0.802)
ALL EVALUABLE PATIENTS
Median OS: P-CAP: 17.7 mos [95% CI (8.5, 24.6)]
Median OS: CAP: 10.9 mos [95% CI (5, 16.9)]
p-value = 0.0161
Hazard ratio: 0.410(0.193, 0.868)
Richards et al., ASCO 2010
Phase III trial (1:1 Cape/Peri vs Cape) started
PTEN Expression and Cetuximab Efficacy
• Fisher’s Exact Test p=0.008
• Concordance primary tumor sample/metastasis: 27/45 (60%)
PTEN +(n=33)
PTEN –(n=22)
Responders (CR+PR+SD) 12 (36%) 1 (5%)
Non-responders 21 (64%) 21 (95%)
Loupakis et al, ASCO 2008Loupakis et al, J Clin Oncol 2009
n=55
1.0
0.8
0.6
0.4
0.2
0.0
0 2.5 5.0 7.5 10.0 12.5 15.0
Months
PF
S e
sti
ma
te
Log-rank test: p=0.005HR=0.49; 95% CI: 0.20–0.75
PTEN + median PFS = 4.7 months
PTEN – median PFS = 3.3 months
CR = complete response; PR= partial response SD = stable disease
PTEN-
PTEN+
PTEN and KRAS Status: Effect on Efficacy
n=45
PTEN + KRAS wild-type median= 5.5 months
All other median PFS= 3.8 months
• Fisher’s Exact Test p=0.0080 2.5 5.0 7.5 10.0 12.5 15.0
Months
PTEN + KRAS wild-type
(n=17)All other (n=28)
Responders (CR+PR+SD) 8 (47%) 1 (4%)
Non responders 9 (53%) 27 (96%)
Log-rank test: p=0.001HR=0.42; 95% CI: 0.17–0.65
1.0
0.8
0.6
0.4
0.2
0.0
PF
S e
sti
ma
te
Loupakis et al, ASCO 2008
PTEN-
PTEN+
Challenges with PTEN
• Expression in primary tumors does not reflect expression in metastases
• PTEN is not mutated in mCRC, its expression can be regulated by methylation, miRNAs, and/or other regulatory mechanisms
• Difficulty in standardizing IHC in different labs
Supplemental Figure 1: Representative examples of PTEN positive (A, B) and negative (C, D) cases. The cases reported in A and C panels were evaluated atOspedale Niguarda Ca’ Granda (Milan, Italy) whereas those in B and D at the Institute of Pathology in Locarno (Switzerland).
Sartore-Bianchi et al, Cancer Res 2009
Potential relationship between KRAS status and response to EGFR monoclonal antibodies, alone or in
combination with irinotecan, in chemorefractory patients
Responds tostandard dose
22%
Responds toincreaseddose ~5%
Non-responder:KRAS mutant 40%
Non-responder:BRAF mutation 10%
Non-responder:Loss of PTEN
or PI3K mutation% unknown
Non-responder:Reason unknown
% unknown
KRASwild-type
KRASmutant
Wong and Cunningham, J Clin Oncol 2008
BRAF Mutations in CRC
• BRAF is primary effector of KRAS signaling
• BRAF mutations: • Occur most frequently
in exon 15 (V600E)• Found in 4%-14% of
patients with CRC• Mutually exclusive
with KRAS mutations
Raf
MEK
Erk
P
P P
P
Tumor cellproliferationand survival
EGF
Tumor Cell
Ras
Yarden. Nat Rev Mol Cell Biol. 2001;2:127; Di Nicolantonio. J Clin Oncol. 2008;26:5705; Artale. J Clin Oncol. 2008;26:4217.
Wild-type BRAF is required for response to EGFR inhibitors in mCRC
Di Nicolantonio et al., J Clin Oncol 2008
BRAF wild-typeBRAF mutant
p=0.0010
Patients with KRAS wild-type status
100
80
60
40
20
0O
S (
%)
0 200 400 600 800 1,000 1,200 1,400
BRAF wild-typeBRAF mutant
p<0.0001
Time since start of treatment (days)
Time since start of treatment (days)
100
80
60
40
20
0
PF
S (
%)
0 100 200 300 400 500 600 700 800 900
CRYSTAL trial update: outcome in KRAS wild-type/ BRAF mutated mCRC
KRAS wt/BRAF wt (n=566)
KRAS wt/BRAF mt (n=59)
FOLFIRI
(n= 289)
FOLFIRI +Cetuximab
(n= 277)
FOLFIRI
(n=33)
FOLFIRI +Cetuximab
(n=26)
mOS (mo) 21.6 25.1 10.3 14.1
HR [95% CI]p-valuea
0.83 [0.687–1.004]0.0549
0.91 [0.507–1.624]0.7440
mPFS (mo) 8.8 10.9 5.6 8.0
HR [95% CI]p-valuea
0.68 [0.533–0.864]0.0016
0.93 [0.425–2.056]0.8656
RR (%)[95% CI]
42.6[36.8–48.5]
61.0[55.0–66.8]
15.2[5.1–31.9]
19.2[6.6–39.4]
p-valueb <0.0001 0.9136
aStratified log-rank test; bCochran-Mantel-Haenszel testVan Cutsem et al, ASCO GI 2010
Bevacizumab: OS independent of biomarker status in first-line mCRC
Overall survival also independent of p53, VEGF (plasma and tissue) and TSP-2
(0.45–1.10)(0.15–0.70)
0.700.32
26.3525.07
99 47
17.4516.26
92 28
191 75
p53 overexpressionPositiveNegative
(0.30–0.95)(0.32–1.42)
0.540.67
27.70NR
76 35
21.7216.36
63 31
139 66
p53 mutation statusMutantWild-type
(0.37–1.20)(0.34–0.82)
0.670.57
19.9127.70
51 68
13.6021.72
37 57
88125
KRAS and BRAF mutation status
MutantWild-type
(0.01–1.06)(0.34–0.82)
0.110.53
15.9326.35
7120
7.9517.45
3 97
10217
BRAF mutation statusMutant
Wild-type
(0.37–1.31)(0.34–0.99)
0.690.58
19.9127.70
44 85
13.60 17.64
34 67
78152
KRAS mutation statusMutant
Wild-type
(0.39–0.85)0.5726.3514717.45120267All subjects
(95% CI)HRMedian
(months)nMedian
(months)nNBiomarker
Placebo + IFL
0.2 0.5 1 2 5
HR
Bev + IFL
Jubb, et al. JCO 2006Ince, et al. JNCI 2005
Bev = bevacizumabTSP-2 = thrombospondin-2
Schneider, et al. J Clin Oncol; 2008
Kaplan-Meier curve for overall survival (OS) in experimental arm by genotype; (A) vascular endothelial growth factor (VEGF)-2578 C/A; (B) VEGF-1154 G/A
VEGF Polymorphisms and Predictive Value in E2100
(Pac +/- Bev Metastatic Breast Cancer)
Caveats: • Predictive for OS, but not PFS• Did not include Pac Rx alone group
Phase III Trial of IFL +/-Bevacizumab in mCRC: PFS
HR=0.54, P<0.00001mPFS: 6.2 vs 10.6 mo
0.2
0 10 20 300
0.8
1.0
0.4
0.6
Months
Pro
po
rtio
n p
rog
res
sio
n-f
ree
IFL + placeboIFL + bevacizumab
Hurwitz et al. N Engl J Med 2004
CRYSTAL trial: FOLFIRI +/- Cetuximab: PFSITT population, independent review
Months
PF
S e
stim
ate
1.0
0.8
0.9
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 2 4 6 8 10 12 14 16 18 20
8.9 mo
8.0 mo
FOLFIRI
FOLFIRI + Cetuximab
Subjects at risk
FOLFIRI alone 599 492 402 293 178 83 35 16 7 4 1Cetuximab + FOLFIRI
599 499 392 298 196 103 58 29 12 5 1Van Cutsem et al. NEJM 2009
HR = 0.851P = 0.0479
RR
46.9%
38.7%
P = 0.0038
George Sledge
Anti-VEGF Therapy: Tool to assess predictive marker
Grade 3/4 Hypertension Is Associated With Improved Median OS in E2100
Median OS:
25.3 mo vs. 38.7 mop=0.002
Schneider et al; J Clin Oncol, 2008
Hypertension is a Biomarker of Efficacy in Patients with Metastatic Renal Cell
Carcinoma Treated with Sunitinib
Brian Rini et al (2010)
“…The primary HTN endpoint was an SBP increase 20 mmHg or DBP increase 10 mmHg within the first 60 days of Tx.
….Conclusions: HTN during Tx does not predict clinical benefit from BV based on PFS or OS.….”
Hurwitz et al. ASCO 2010
Analysis of early hypertension and clinical outcome with bevacizumab
Cytokine increase on BEV therapy
Kopetz et al., JCO 2010
Multiple Potentially Active Biologics• Anti-VEGF
• bevacizumab• VEGF-Trap• vatalanib• sorafenib• Sunitinib• cediranib• axitinib• IMC1121b
• Anti-EGF-R• cetuximab• Panitumumab• erlotinib• gefitinib• lapatinib
• Further targets (selection)• IGF-R• mTOR• PKC• PI3K/AKT• Dll4/Notch• Hedgehog• JAK/Stat• C-met• MEK• TRAIL• MMPs• CDKs
Non-Specificity of VEGFR Kinase Inhibitors- The Human Kinome Tree -
• The larger the red circle, the more effective the drug is for the target
• Markers that may be predictive for one TKI, may not be predictive for another
Karaman et al., Nature Biotechnol. 2008
Bevacizumab vs EGFR Antibodies in Advanced CRC - Simplified
Agent Strength Weakness
Bevacizumab • Delay in tumor progression
• Gain in time• Toxicity profile
• No single agent activity
• Weak effect on RR
EGFR antibodies
• Single agent activity• Consistent increase
in RR• Activity independent
of line of therapy• Predictive marker
• Gain in time to progression moderate
• Toxicity profile
Conclusions
• CRC has morphed into 2 distinct entities:• KRAS wt and KRAS mut CRC• No EGFR therapy in KRAS mut CRC!
• If response is primary goal (symptoms, conversion therapy), cetuximab is reasonable first-line option in KRAS wt CRC
• In palliative situation, BEV-containing regimen remains preferable, even in KRAS wt CRC
• More impressive gain in time, less toxicity• Treat to progression (and perhaps beyond?)
• Dual antibodies not outside clinical trials in first-line therapy
• Might be different in salvage scenario
• We need new drugs in CRC – and KRAS mut CRC can be the target for new drug development
Adjuvant Therapyof Colon Cancer
National Comprehensive Cancer Network (NCCN), 2008; Greene et al., 2002.AJCC = American Joint Committee on Cancer.
AJCCv6 TNM Staging Definitions
Primary tumor (T) Tis Carcinoma in situ
T1 Tumor invades submucosa
T2 Tumor invades muscularis propria
T3 Tumor invades through muscularis propria or subserosa
T4 Tumor directly invades other organs or structures
Regional lymph nodes (N) N0 No regional lymph node metastases
N1 Metastases in 1–3 regional lymph nodes
N2 Metastases in 4 or more regional lymph nodes
Distant metastases (M) M0 No distant metastases
M1 Distant metastases
T4a: perf. visceral peritoneumT4b: invasion of organs
N1a: 1 N+N1b: 2-3 N+
N2a: 4-6 N+N2b: >7 N+
TNM / AJCC v7 Effective Jan 2010
AJCC v7
Gunderson et al, JCO 2009
Stage II Stage III
Beyond 5-FU in the adjuvant setting
Completed studies:• Capecitabine (X-ACT) – non-inferior to 5-FU
• Oxaliplatin (MOSAIC, NSABP C-07, XELOXA)
• Irinotecan (CALGB 89803, ACCORD-2, PETACC-3)
• Bevacizumab (NSABP C-08, AVANT)
• Cetuximab in KRAS wt CC (N1047)
Ongoing studies:• Bevacizumab (QUASAR-2, E5202)
• Cetuximab in KRAS wt CC (PETACC-8)
Adjuvant Trials in Colon Cancer with Bevacizumab
Stage II/III colon cancer (N=3450)
XELOX 6m +Bevacizumab 12m
FOLFOX4 6m +Bevacizumab 12m
AVANT FOLFOX4 6m
Stage II/III colon cancer (N=2710)
25% Stage II
mFOLFOX6 6m
mFOLFOX6 6m +Bevacizumab 12m
NSABP C-08
Reported at ASCO2009
NSABP C-08
R
mFOLFOX6 q2wk X 6 mo
BEV* q2wk X 1 yr
*5mg/kgN=2710 pts25% stage II Wolmark et al ASCO 2009
NSABP C-08 – DFS
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
020
4060
8010
0
Ev 3yDFSmFF6+B 291 77.4mFF6 312 75.5
HR 0.89P 0.15
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
6070
8090
100 NSABP C-08 – DFS
Ev 3yDFSmFF6+B 291 77.4mFF6 312 75.5
HR 0.89P 0.15
NSABP C-08 HR over Time
0.0004
0.0040.02
0.05 0.08
AVANT Adjuvant Colon Cancer Study
n=3451 Stage III or high-risk stage II
colon cancer
FOLFOX4 q2wk
Bev 7.5 mg/kg q3wk
24 Weeks 48 Weeks
Stratified bystage and
region
1:1:1
Bev 5 mg/kg, q2wk
XELOX q3wk
• 3451 patients were enrolled between November 2004 and June 2007• Primary analysis: compare DFS between control and each treatment
arm in stage III patients
FOLFOX4 q2wk
Bevacizumab 7.5 mg/kg q3wk
Safety data presented at ESMO September 2009 by P. Hoff
Revised, Biomarker-driven Design of N0147
Stage 3 Colon Cancer
(N = 3768)
PREREGISTER
RANDOMIZECentralized
K-ras analysis
KRAS WT
KRAS Mut
REGISTER
Arm G
• Adjuvant therapy per primary oncologist
• Report therapy given
• Annual status through year 8
Arm AmFOLFOX6
Arm DmFOLFOX6 +
Cetuximab
Alberts/Goldberg et al., ASCO 2010
0
10
20
30
40
50
60
70
80
90
100
0 6 12 18 24 30 36
Time (Months)
%Al
ive
and
Dis
ease
Fre
e
FOLFOX
FOLFOX + Cmab
Phase III Trial N0147 - FOLFOX +/- Cetuximab by KRAS status in Stage III Colon Cancer: DFS
KRAS WT KRAS Mut
Arm 3 Year Rates (95% CI)
HR (95% CI)
P-value
FOLFOXN=374
67.2%(61.4-73.5%)
1.2(0.9-1.6)
0.13
FOLFOX + CmabN=343
64.2%(58.7-70.2%)
0
10
20
30
40
50
60
70
80
90
100
0 6 12 18 24 30 36
Time (Months)
% A
live
and
Dis
ease
Fre
e
FOLFOXFOLFOX + Cmab
Arm 3 Year Rates (95% CI)
HR (95% CI)
P-value
FOLFOXN=902
75.8%(72.1-79.6%)
1.2(0.96-1.5)
0.22
FOLFOX + CmabN=945
72.3%(68.5-76.4%)
Alberts/Goldberg et al., ASCO 2010
Reason For Failure of Biomarker-driven Adjuvant Trial in Colon Cancer: EMT?
Loss of epithelial and gain of mesenchymal markers
E-cadherinCytokeratinLaminin-1
MUC-1EGFR
N-cadherinVimentin
FibronectinETS
a-SMA
Epithelial phenotype Mesenchymal phenotype
Carcinoma in situ EMT Hematogenous dissemination
Metastasis
Kalluri & Weinberg, J Clin Invest 2009
Should patients with stage II colon cancer receive adjuvant therapy?
% o
f P
atie
nts
QUASAR: OS in patients with “no clear indication for chemo” (mostly stage II)
5-FU/LV vs surgery alone
P = .025-year OS, Observation = 77.4% vs Chemotherapy = 80.3%Relative risk = 0.83 (95% CI, 0.71-0.97)
Years
QUASAR group, Lancet 2007
0 1 2 3 4 5 6 7 8 9 100
20
40
60
80
100Observation (n=1622)
Chemotherapy (n=1617)
5-yr OS difference: 2.9%
“High-risk” Stage II Colon Cancer
• Clinical-pathological parameters• T4 tumors• Less than 10 (12) LNs examined• Obstruction/perforation• Lymphatic or vascular invasion• Undifferentiated histology
• Molecular parameters• Single marker vs signature - TBD
Defective MMR - Colon cancer
• Characterized by presence of MSI & loss of MLH1, MSH2, MSH6 or PMS2 expression
• ~15% of Sporadic CC, >90% loss of MLH1
• Clinical Correlations: Right sided, Female, Early stage, Better prognosis
• Tumors: Poorly differentiated, Signet-ring-cell, Lymphocytic infiltration, near diploid
• MMR-D cells resistant to 5-FU1,2
1Carethers, 1999; 2Arnold 2003
Mismatch Repair Deficiency (MMR-D):Unique Biological Subgroup of Colon Cancer
Imai and Yamamoto. Carcinogenesis 2008Umetani, Annals of Surgical Oncology 2000
Rosen et al. Modern Pathology (2006) 19, 1414-1420
PCR on tumor DNA for MSI
(microsatellite instability)
IHC for MMR protein status
MLH1+
MSH2+MLH1-
MSH2-
Thus, IHC for MMR proteins and PCR for MSI detect two manifestations of the same tumor biology:
• MMR-D is synonymous with MSI-H• MMR-P is synonymous with MSI-L/MSS
DFS/OS in Stage II MMR-D Patients(N=102)
HR: 2.80 (0.98-8.97)p=0.05
5-yr DFS
Untreated 87%Treated 72%
Sargent JCO 2010
HR: 3.15 (1.07-9.29)p=0.03
Untreated 93%Treated 75%
5-yr OSN = 55N = 47
PETACC 3: Multivariate Analysis Prognostic Factors Stage II
Roth AD, et al. ASCO 2009, JCO 2010.
Markers HR [95% CI] P value
T4 v. T3 2.58 [1.56 - 4.28] 0.00024
MSI-H v. MSS 0.28 [0.10 - 0.72] 0.0089
18qLOH 1.37 [0.67 - 2.77] 0.38
Colon Cancer Technical Feasibility
Development StudiesSurgery Alone
NSABP C-01/C-02 (n=270)
CCF (n = 765)
Selection of Final Gene List & Algorithm
Development Studies Surgery + 5FU/LV
NSABP C-04 (n=308)
NSABP C-06 (n=508)
Clinical Validation Study – Stage II Colon Cancer
QUASAR (n>1200)
Test prognostic, but not predictive!
Genomic Health: Development and Validation of an 18-Gene RT-PCR Colon Cancer Assay
Validation of Analytical Methods
761 genes
375 genes
18 genes
ASCO 2009
Kerr et al., ASCO 2009, abstr. 4000
QUASAR RESULTS: Recurrence Score, T Stage, and MMR Deficiency are Independent Predictors of Recurrence in
Stage II Colon Cancer
Multivariate AnalysisKerr et al., ASCO 2009, abstr. 4000
QUASAR Results: Recurrence Score, T Stage, and MMR Deficiency are Key
Independent Predictors of Recurrence in Stage II Colon Cancer
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
0 10 20 30 40 50 60 70
Recurrence Score
Ris
k o
f re
curr
en
ce
at
3 y
ears
T3 and MMR deficient (11%)
T4 stage (13%)
T3 and MMR proficient (76%)
Kerr et al., ASCO 2009, abstr. 4000
Decision Algorithm in Adjuvant Therapy
Resected Colon Ca
Stage II Stage III
FOLFOXXELOX
High-Risk
dMMR
No therapy!5-FU/LV or
Capecitabine*
*
*pts not considered candidates for oxaliplatin
T4 and/or<12 LNs
Low-Risk
Intermed. Risk
yes
yes
no
no
?Marker signature?
Grothey, Oncology 2010
How A Cell Works
