- The document discusses biomarkers such as KRAS, NRAS, BRAF and their roles in predicting response to targeted therapies for metastatic colorectal cancer. - The PRIME study showed that testing for additional RAS mutations beyond KRAS exon 2 identified more patients unlikely to benefit from anti-EGFR therapy. Around 17% of mCRC patients had non-KRAS exon 2 RAS mutations. - The FIRE-3 study found that for patients with wild-type RAS, frontline FOLFIRI plus cetuximab resulted in improved progression-free survival and overall survival compared to FOLFIRI plus bevacizumab. Testing for all RAS mutations is

1. Department of GI Medical Oncology
ALPHABET SOUP: MAKING SENSE
OF KRAS, BRAF, RAS AND OTHER
BIOMARKERS IN METASTATIC
COLORECTAL CANCER
Cathy Eng, M.D., F.A.C.P.
Associate Professor
Associate Medical Director, Colorectal Center
Director of Network Clinical Research, GI Med Oncology
Co-Chairman, SWOG Rectal Subcommittee
April 23, 2014

2. Cancers of the Colon and Rectum
International Statistics
Jemal et al: Cancer Epidemiol Biomarkers Prev; 19(8) August 2010; Siegel et al: CA Cancer J Clin 2014
Incidence
Mortality
1.2 Million
609,000
Worldwide
per annum
USA (2014)
Incidence
Mortality
136,830
50,310
Colorectal cancer is the 3rd most
common cancer in
men and the 2nd in women.

3. Advances in the Treatment
of Metastatic Colorectal Cancer
1980 1985 1990 1995 2000 2005
Therapeutic concepts
Palliative CT
Neoadjuvant CT
Capecitabine
Oxaliplatin
Cetuximab
Bevacizumab
Irinotecan
5-FU
Panitumumab
Targeted therapies
{
5-FU = 5-fluorouracil; CT = chemotherapy.
{Cytotoxic chemotherapies
Ras
OS: 20M
OS: 32 months
Aflibercept
Regorafenib

4. 15.6
20.3 19.9
21.3
23.1
28
17.6
19.2
0
5
10
15
20
25
30
First-Line Bevacizumab in mCRC:
Overall Survival
*P<0.001; †P = 0.0769.
1. Hurwitz H et al. N Engl J Med. 2004;350:2335-2342; 2. Saltz LB et al. J Clin Oncol. 2008;26:2013-2019;
3. Fuchs C et al. J Clin Oncol. 2007;25:4779-4786; 4. Fuchs C et al. J Clin Oncol. 2008;26:689-690;
OS(months)
*
NO169662
AVF2107g1
BICC-C3,4

5. Approved Anti-VEGF Agents
Antiangiogenic
agent
Description Target Approval
Bevacizumab
Recombinant
humanized
monoclonal antibody
VEGF-A
1st-line
mCRC1,2:
•FDA 2004
•EMEA 2005
2nd-line
mCRC1:
•FDA 2006,
2013
Aflibercept
Fully human fusion
protein
VEGF-A
VEGF-B
PIGF
2nd-line
mCRC3,4:
•FDA 2012
•EMEA 2013,
•TGA 2013
Regorafinib Small molecule TKI
VEGFR-1,2 & 3 PDGFR-b,
TIE-2, FGFR-1, Ret, Kit, & Raf
kinases
Salvage5,6:
•FDA 2012
•CHMP 2013
•TGA 2013
CHMP, Committee for Health and Medicine Products; EMEA, European Medicines Agency; FDA, United States Federal Drug
Administration, FGFR, fibroblast growth factor receptor; PDGFR, platelet-derived growth factor receptor; PlGF, placental growth
factor; TKI, tyrosine kinase inhibitor; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor
.

6. Biomarker Development
 Review of Definitions:
 Prognostic marker
 Independent of treatment
 May impact surveillance
 Predictive marker
 Impacts type of treatment provided

7. Molecular Markers for Anti-VEGF
 None identified and validated:
 Bevacizumab
 Aflibercept
 Regorafenib
 Anti-EGFR Therapy
 Predictive: KRAS/NRAS
 Prognostic: BRAF

8. Current Molecular Markers

9. KRAS
 Proto-oncogene
 First globally utilized predictive marker for
the treatment of MCRC when considering
anti-EGFR therapy
 30%-50% of all patients
 MT (exon 2): codons 12, 13, 61, and
rarely 146
 KRAS WT does = efficacy of therapy nor
does it indicate duration of response

10. Copyright © American Society of Clinical Oncology
Khambata-Ford, S. et al. J Clin Oncol; 25:3230-3237 2007
Cetuximab and K-ras modulate signaling through the
epidermal growth factor receptor (EGFR) pathway

11. BRAF MT
 Serine-threonine kinase belong to the RAF
family
 Mutation also leads to constitutive activation
 V600E accounts for 90% of mutations
 Found in < 10 % of all CRC patients
 Associated with hypermethylation of CpG
island.
 Mutually exclusive with KRAS MT
 Prognostic but NOT predictive
 All studies insufficiently powered to provide
sufficient data to determine use of anti-EGFR
therapy based on BRAF status.

12. NRAS
 Resembles Kras
 Oncogene
 < 5% of all mCRC
 Mutations in codons 12, 13, 61, 117 and
146
 Usually codon 61
 Mutually exclusive with KRAS

13. Front-line chemotherapy with anti-EGFR
therapy

14. Update on PRIME Study Phase III
Douillard JY, et al. J Clin Oncol. 2010;28:4697-4705.
Patients
• Previously untreated mCRC
• Fluorouracil-based adjuvant
chemotherapy allowed if PD
occurred
≥6 mo after completion; no
oxaliplatin
• Tumor tissue from primary
tumor or metastasis available
for biomarker analysis
• ECOG PS 0-2
• N=1183
Primary endpoint: PFS
Panitumumab 6.0 mg/kg q 2 wk
FOLFOX4 q 2 wk
1:1 Randomization
FOLFOX4 q 2 wk

15. Distribution of mutations in mCRC
RAS wt
~50%
KRAS mt
(exon 2)
~40%
KRAS mt
(non exon 2
KRAS mt) &
NRAS mt
~10%
Rare KRAS Mutations
NRAS Mutations
Douillard JY. ASCO 2013. Abstract 3620; Oliner KS. ASCO 2013. Abstract 3511.

16. PRIME Biomarker Analysis: Analysis of
KRAS/NRAS and BRAF Mutations
RAS and BRAF Status FOLFOX4 Alone Panitumumab + FOLFOX4
KRAS exon 2 (codon 12/13)
WT
MT
331
219
325
221
WT KRAS exon 2 tumors tested for RAS and BRAF (n = 321) (n = 320)
WT KRAS exon 2/MT other RAS, n (%) 57 (18) 51 (16)
KRAS exon 3 (codon 61), n (%)
WT
MT
Failure
306 (95)
14 (4)
1 (0)
308 (96)
10 (3)
2 (1)
KRAS exon 4 (codons 117/146), n (%)
WT
MT
Failure
296 (92)
15 (5)
10 (3)
288 (90)
21 (7)
11 (3)
NRAS exon 2 (codons 12/13), n (%)
WT
MT
Failure
307 (96)
14 (4)
0 (0)
308 (96)
8 (3)
4 (1)
NRAS exon 3 (codon 61), n (%)
WT
MT
Failure
305 (95)
14 (4)
2 (1)
305 (95)
12 (4)
3 (1)
NRAS exon 4 (codons 117/146), n (%)
WT
MT
Failure
313 (98)
0 (0)
8 (2)
316 (99)
0 (0)
4 (1)
BRAF exon 15 (codon 600), n (%)
WT
MT
Failure
280 (87)
29 (9)
12 (4)
286 (89)
24 (8)
10 (3)
Oliner J, et al. J Clin Oncol. 2013;31(Suppl): Abstract 3511. Oliner J, et al. Eur J Cancer. 2013;49(Suppl 2): Abstract 2275.

17. Revised PRIME Consort Diagram
Douillard et al: NEJM, 2013

18. PRIME: Progression-free survival in patients with (A) Original wild-type
(WT) KRAS, (B) Updated All WT RAS, Overall survival in patients with (C)
Original WT KRAS and (D) All WT KRAS
Douillard J et al. JCO 2010;28:4697-4705; NEJM, 2013
©2010 by American Society of Clinical Oncology
D
B

19. PFS: Wild-Type (WT) KRAS Exon 2 + mutant
(MT) Other RAS
Oliner KS. ASCO 2013. Abstract 3511.

20. OS: Wild-Type (WT) KRAS Exon 2 + mutant
(MT) Other RAS
Oliner KS. ASCO 2013. Abstract 3511.

21. PRIME: Summaryand
ClinicalImplications
 About 17% of patients with mCRC harbor mutations
beyond KRAS exon 2 mutations
 Excluding patients with RAS mutations identifies
patients more likely to benefit from anti-EGFR therapy.
 Practical interpretation: until an all-RAS test becomes
available, EGFR monoclonal antibodies have the
potential to be detrimental in patients who may harbor
an unrecognized RAS mutation when administered with
oxaliplatin-based chemotherapy regimens
Douillard JY. ASCO 2013. Abstract 3620; Oliner KS. ASCO 2013. Abstract 3511.

22. Treatment choice: Front line
chemotherapy with anti-EGFR therapy
or anti-VEGF therapy?

23. PEAK: Randomized Phase II (KRAS WT)
FOLFOX/Pmab
(N=142)
FOLFOX/Bev
(N=143)
Median PFS (95% CI) 10.9 (9.4-13.0) 10.1 (9.0-12.6)
Median OS (95% CI) 34.2 (26.6-NR) 24.3 (21.0-29.2)
ORR (95% CI) 58 (49-66) 54 (45-62)]
Subsequent therapy:
Anti EGFR
21% 38%
Anti-VEGF 40% 24%
Schwarzberg et al: JCO 2014

24. PEAK: Randomized Phase II (KRAS WT
and rare RAS WT)
FOLFOX/Pmab
(N=88)
FOLFOX/Bev
(N=82)
Median PFS (95% CI) 13.0 (10.9-15.1) 9.5 (9.0-12.7)
Median OS (95% CI) 41.3 (28.8-41.3) 28.9 (23.9-31.3)
ORR (95% CI) 64 (52.7-73.6) 61 (49-71.2)
Subsequent therapy:
Anti EGFR
22% 37%
Anti-VEGF 40% 33%
Schwarzberg et al: JCO 2014

25. FIRE-3 Phase III Study Design
Heinemann V. ASCO 2013. Abstract LBA3506.
Patients
• mCRC
• KRAS wild-type
• ECOG PS 0-2
• 1st line therapy; prior
adjuvant chemotherapy
allowed if completed
>6 mo before inclusion
• N=592 Primary Endpoint: Response Rate
FOLFIRI + Cetuximab
(Cetuximab: 400 mg/m2 loading dose;
250 mg/m2 weekly)
1:1 Randomization
FOLFIRI + Bevacizumab
(Bev: 5 mg/kg every 2 weeks)

26. FIRE-3: Overall Response Rate
Endpoint
FOLFIRI +
Cetuximab
FOLFIRI +
Bevacizumab
OR P Value
ORR, intent-to-treat
(ITT) population
(N=592)
62.0% 58.0%
1.18
(0.85-1.64)
0.183
Complete response 4.4% 1.4%
Partial response 57.6% 56.6%
Stable disease 17.5% 28.8%
Progressive disease 7.1% 5.4%
Not evaluable 13.1% 7.8%
ORR, Evaluable (N=526) 72.2% 63.1%
1.52
(1.05-2.19)
0.017
Heinemann V. ASCO 2013. Abstract LBA3506.

27. FIRE-3: Progression Free Survival
Stintzing S. ASCO 2013. Abstract LBA3506

28. FIRE-3: Overall Survival
Heinemann V. ASCO 2013. Abstract LBA3506.

29. Consort FIRE-3 Diagram
N=592
KRAS exon 2 wild-type
ITT population
N=407 (69%)
RAS evaluable population
N=65 (16%)
‘New’ RAS mutant
N=342
RAS wild-type
N= 171
FOLFIRI +
Cetuximab
N= 34
FOLFIRI
Cetuximab
N= 171
FOLFIRI +
Bevacizumab
N= 31
FOLFIRI +
Bevacizumab
N=752
mCRC 1st-line
unselected patients
N=58
FOLFIRI +
Cetuximab
N=55
FOLFIRI +
Bevacizumab
N=113
KRAS exon 2 mutant
population*
KRAS unknown= 30
No treatment= 13
No treatment KRAS
mt = 4
Stinzing et al: ESMO, 2013

30. KRAS Wildtype Exon 2 Additional Subsets
?
? ?
EXON 1 EXON 2 EXON 3 EXON 4
EXON 2 EXON 3 EXON 4
KRAS
NRAS
12 13
12 13
61 146
59 61 117 146
wt
? ?
EXON 1
EXON 15EXON 11BRAF
600
?
?
Heinemann V, et al. J Clin Oncol. 2013;31(Suppl): Abstract LBA3506.

31. Events
n/N (%)
Median
(months)
95% CI
― FOLFIRI + Cetuximab 91/171
(53.2%)
33.1 24.5 – 39.4
― FOLFIRI + Bevacizumab 110/171
(64.3%)
25.6 22.7 – 28.6
HR 0.70 (95% CI: 0.53 – 0.92)
p (log-rank)= 0.011
FIRE-3: Overall survival RAS* all wild-type
0.0
12 24 36 48 60 72
months since start of treatment
171
171
No. at
risk
128
127
71
68
39
26
20
9
6
1
0.75
1.0
0.50
0.25
0.0
Probabilityofsurvival
Δ = 7.5 months
* KRAS and NRAS exon 2, 3 and 4 wild-typeStinzing et al: ESMO, 2013

32. FIRE-3 Update:Overall Survival by
All-RASMutationStatus
Study Population
FOLFIRI +
Cetuximab
FOLFIRI +
Bevacizumab
HR
P
Value
ITT (N=592) 28.7 months 25.0 months 0.77 0.017
RAS WT (n=342) 33.1 months 25.6 months 0.70 0.011
RAS MT (n=65) 16.4 months 20.6 months 1.20 0.57
BRAF MT (n=48) 12.3 months 13.7 months 0.87 0.65
Stintzing S. European Cancer Conference 2013. Abstract LBA17.

33. FIRE-3: Summary and Clinical Implications
 Current data limitations
 No central assessment of response
 OS data continues to mature
 Duration of second and subsequent lines of therapy
not reported
 Practical impact
 EGFR antibodies added to FOLFIRI can be
considered a viable option in first-line, KRAS wild-
type mCRC
 Next steps
 CALGB 80405 data (in 2014) may clarify results
Heinemann V. ASCO 2013. Abstract LBA3506.

34. Should all RAS WT patients
receive anti-EGFR therapy
front-line?

35. New EPOC Study: Chemotherapy
± Cetuximab in Operable KRAS-WT mCRC
 Original EPOC study showed 8% PFS benefit to addition of neoadjuvant
FOLFOX to surgery in mCRC patients with operable liver metastases[1]
 New EPOC study evaluated addition of cetuximab to standard neoadjuvant
chemotherapy in mCRC[2]
 Primary endpoint: PFS
 Secondary endpoints: OS, preop response, pathologic resection status,
periop safety, QoL, cost-effectiveness
Patients with
resectable KRAS WT
mCRC with liver mets
(N = 621)
Neoadjuvant Chemotherapy*
(randomized n = 134;
primary analysis n = 116)
Neoadjuvant Chemotherapy*
+ Cetuximab
(randomized n = 137;
N = 117)
1. Nordlinger G, et al. Lancet. 2008. 2. Primrose JN, et al. ASCO 2013. Abstract 3504.
*CAPOX, OxMdG, IrMdG

36. New EPOC: Neoadjuvant Chemotherapy ±
Cetuximab in Operable KRAS-WT mCRC: PFS
 Median PFS
significantly
worse with
cetuximab: 14.1
months vs 20.5
months with
chemotherapy
alone
 Study stopped at
predefined futility
analysis
 Immature data, but
more events
unlikely to change
result
Primrose JN, et al. ASCO 2013. Abstract 3504.
Proportionprogressionfree
1.00
0.75
0.50
0.25
0.00
0 6 12 18 24 30 36 42 48 54 60
Time to progression or death (months)
HR: 1.49 (95% CI: 1.04-2.12); P = .030
Number at risk
Chemo alone
Chemo + Cetuximab
116
117
89
87
65
54
38
24
23
15
12
5
5
3
2
2
1
1
1
0
0
0
Chemo alone
Chemo + cetuximab

37. Why did the new EPOCH study fail?
 KRAS is a predictive marker of potential benefit
for the use of EGFR inhibition.
 Cetuximab does not have a role in the adjuvant
setting
 N0147: FOLFOX +/- cetuximab failed to demonstrate
an improvement in DFS in stage III colon cancer
 3-yr DFS: 74.6% vs 71.5% with the addition of
cetuximab (HR, 1.21; 95% CI, 0.98–1.49; P=.08)
 Is it the combination of FOLFOX and
cetuximab?
Alberts et al: JAMA. Apr 4, 2012; 307(13): 1383–1393.

38. Upcoming: Liver-Only Trials

39. BOS-2 (EORTC 40091): Phase II
KRAS WT Resectable Liver Mets
R
A
N
D
O
M
I
Z
E
FOLFOX
• First-line
mCRC
• N=360
FOLFOX + bevacizumab
FOLFOX + panitumumab
Study amended: Wild-type KRAS tumors only
Primary Endpoint: PFS
http://www.clinicaltrials.gov/ct2/show/NCT01508000?term=BOS-2&rank=1

40. BOS -3 (EORTC-1207) Phase II/III Study
Design (Pending)
http://www.clinicaltrials.gov/ct2/show/NCT01646554?term=BOS-2&rank=2
Patients
• mCRC
• KRAS MT
• ECOG PS 0-1
• 1st line therapy; prior
adjuvant chemotherapy
allowed if completed
>12 mo before inclusion
Primary endpoint: PFS
FOLFOX + Aflibercept
(Aflibercept: 4 mg/m2)
1:1 Randomization
FOLFOX

41. CALGB/SWOG 80405:
Results: ASCO 2014
R
A
N
D
O
M
I
Z
E
FOLFOX or FOLFIRI* + cetuximab
• First-line
mCRC
• Amended
accrual;
N=2300
wild-type
patients
FOLFOX or FOLFIRI* +
cetuximab + bevacizumab
FOLFOX or FOLFIRI* +
bevacizumab
Study amended: Wild-type KRAS tumors only
Primary endpoint: OS

42. SWOG 1406: BRAF MT mCRC
PI: S Kopetz

43. Conclusions:
 All RAS WT tumor types may provide
more benefit in OS if an anti-EGFR
therapy is provided in the front-line setting.
 Provision of anti-EGFR therapy in the
setting of a RAS MT can be detrimental
 Many institutions utilize outside sites for tissue
processing
 Not all codons are identified
 Need a readily available panel with all RAS
mutations