1. Comparison of molecular and pathologic features of stage II and stage III colon cancer in 4 large studies conducted for development of the 12-gene colon cancer Recurrence Score®
Michael J. O’Connell1, Ian C. Lavery2, Richard Gray3, Philip Quirke4, David Kerr5, Margarita Lopatin6, Greg Yothers1, Steven Shak6, Kim Clark-Langone6, Mark Lee6, Norman Wolmark1
1. National Surgical Adjuvant Breast and Bowel Project, Pittsburgh, PA; 2. Cleveland Clinic, Cleveland, OH; 3. Birmingham Clinical Trials Unit, Birmingham, UK; 4. Leeds Institute of Molecular Medicine, Leeds, UK; 5. University of Oxford, Oxford, UK & SIDRA, Qatar; 6. Genomic Health, Inc., Redwood City, CA

2. Disclosures M. J. O’Connell None
I. C. Lavery Honorarium, Educational grant, Genomic Health, Inc.
R. Gray Educational grant, Genomic Health, Inc.
P. Quirke Educational grant, Genomic Health, Inc.
D. Kerr Educational grant, Genomic Health, Inc.
M. Lopatin Employee, Genomic Health, Inc.
G. Yothers Honorarium, Genomic Health, Inc.
S. Shak Employee, Genomic Health, Inc.
K. Clark-Langone Employee, Genomic Health, Inc.
M. Lee Employee, Genomic Health, Inc.
N. Wolmark None

3. Are Stage II and Stage III Colon Cancer Different Diseases?
Implications
Clinical management
Design of therapeutic trials
Challenges
Few studies sufficiently powered to examine stage by covariate interactions
Clinical significance of observed differences

4. Interaction of Stage with Treatment: Data from NSABP Trials
Pooled analysis of NSABP C-01, C-02, C-03, C-04 showed no evidence of treatment by stage interaction*
No evidence of treatment by stage interaction in NSABP C-07**
* Mamounas 1999 JCO 17:1349
** Kuebler 2007 JCO 25:2198

5. PETACC-3 Molecular Study Prognosis (RFS): Univariate Analysis
Marker
Stage II (n=420)
Stage III (n=984)
Interaction HR
p val
MSI (Hi vs Stable)
0.3
0.004
0.7
0.06
0.04
18qLOH
2.1
0.03 1
0.91
0.05
SMAD4 (any loss)
1.4
0.21
1.6
<0.0001
0.23
hTERT (High)
0.32
1.5
0.01
0.92
p53 (High)
1.0
0.98
1.3
0.37
TS (High)
0.5
0.02
0.30
Adapted from Roth, et al. ASCO 2009

6. ACCENT: Differences in Survival Following Recurrence (Stage II vs III)
O’Connell 2009 JCO 26:2336

7. Large Genomic, Pathology, and Clinical Dataset for Development of the Recurrence Score
Colon Cancer Technical Feasibility
Selection of Final Gene List & Algorithm
Clinical Validation Study – Stage II Colon Cancer
QUASAR (n=1,436)
Test Prognosis and Treatment Benefit
Standardization and Validation of Analytical Methods
Development Studies
Stage II/III Surgery Alone
NSABP C-01/C-02 (n=270)
Cleveland Clinic (n = 765)
Development Studies
Stage II/III Surgery + 5FU/LV
NSABP C-04 (n=308)
NSABP C-06 (n=508)
Kerr et al. ASCO 2009, #4000

8. The 12-Gene Colon Cancer Recurrence Score (RS) Predicts Recurrence Following Surgery in Stage II Colon Cancer
Risk of Recurrence at 3 years 0% 5%
10%
15%
20%
25%
30%
35%
Recurrence Score 10 20 30 40 50 60 70 |
Prospectively-Defined Primary Analysis in Stage II Colon Cancer (n=711)
RECURRENCE SCORE
STROMAL
FAP
INHBA
BGN
CELL CYCLE
Ki-67
C-MYC
MYBL2
GADD45B
REFERENCE
ATP5E
GPX1
PGK1
UBB
VDAC2
p=0.004
RS = x Stromal Group
x Cell Cycle Group
x GADD45B
Kerr et al., ASCO 2009, #4000

9. Objective
Examine stage II and stage III colon cancer for pathologic markers and expression of 375 cancer-related genes, including the 12-gene Recurrence Score, and determine
Distribution of markers
Relationship of markers to clinical outcome

10. Methods
634 stage II patients with ≥ 12 nodes examined were compared to 844 stage III patients across 4 independent studies
Focus on ≥ 12 nodes examined to minimize risk of understaging in stage II
Study
Treatment
Stage II
Stage III
NSABP C-01/C-02
Surgery alone 62
139
NSABP C-04
Surgery +5FU/LV 66
171
NSABP C-06
119
273
Cleveland Clinic
387
261

11. Methods
Pathology: MMR (IHC), T stage, tumor grade, mucinous type, nodal status
Gene expression: Reference-normalized expression of 375 cancer-related genes by quantitative RT-PCR from manually micro-dissected paraffin-embedded primary colon tumor tissue
Includes 12 genes from the colon cancer Recurrence Score
Stage-specific associations with recurrence were evaluated by Cox regression, stratified by study

12. Results: Nodal Status Strongly Predicts Recurrence
Surgery Alone
Stage II
Stage III
Proportion Event Free
0.2
0.4
0.6
0.8
1.0
Years 1 2 3 4 5 6 7 8 9 10
Surgery + 5FU 1 2 3 4 5 6 7 8 9 10
Stage II
Stage III
0.2
0.4
0.6
0.8
1.0
Years
HR for Stage III vs. II = 2.86
95% CI , p<0.001
HR for Stage III vs. II = 3.07
95% CI , p<0.001

13. Distribution of Pathologic Markers: Stage II vs Stage III
p=ns
p=0.038
p=0.007
p=ns

14. Pathologic Markers and Recurrence Risk: Interaction with Stage
Interaction of stage and covariate HR 1 2 3 4
Grade
MMR
Mucinous
T Stage
Stage II
Stage III
p = 0.11
p = 0.07
p = 0.005
p = 0.11

15. Concordance of Mean Expression (CT) of 375 Cancer-Related Genes by Stage14
Small absolute differences in mean expression of individual genes in stage II vs. III: median 0.09 CT max 0.58 CT
Only 5 of 375 genes had significant (p<0.05) differences in all 4 studies 2 4 6 8 10 12
Stage II CT 2 4 6 8 10 12 14
CT Stage III

16. Gene Expression and Recurrence Risk: Interaction with Stage
   
HR per SD in Stage II
0.0
0.5
1.0
1.5
2.0
HR per SD in Stage III
45 of 375 genes showed some evidence of interaction with stage (p<0.1)
Expect 37 false positives by chance alone
Interaction p<0.1
No significant interaction

17. Genes/Pathways with Stage-Specific Differences
Mean expression by stage:
Metabolism: FABP4, SI
Invasion: STMY3
Angiogenesis and migration: EFNB2, Maspin
Interaction of gene expression and stage*
MMR-associated
Wnt pathway
Signal transduction
Proteases/protein degradation
Angiogenesis and migration
Immune response
* 37 of 45 genes expected to be false positives

18. Average Distance Between Clusters Average Distance Between Clusters
Similar Coexpression of 48 Recurrence Risk Genes Stage II vs Stage III Colon Cancer
UMPS
MYBL2
CSEL1
CMYC
NME1
HNRPD
SKP2
RRM1
MCM2
RRM2
KI_67
CDC20
P16_INK4
P14ARF
KLK6
GRB10
TGFBI
LAMC2
P21
CDC42BPA
HSPA1A
S100A4
OPN__OSTEOPONTIN
PAI1
GADD45B
EGR1
STMY3
DLC1
IGFBP3
SFRP4
PDGFC
IGFBP7
CALD1
TIMP3
TGFB3
SFRP2
INHBA
FAP
CTHRC1
LOXL2
SPARC
COL1A1
BGN
TIMP2
ANTXR1
ITGB1
AKT3
AKAP12
Average Distance Between Clusters
0.0
0.2
0.4
0.6
0.8
1.0
1.2
HNRPD
UMPS
NME1
MYBL2
CSEL1
CMYC
SKP2
RRM1
MCM2
RRM2
KI_67
CDC20
P16_INK4
P14ARF
GRB10
HSPA1A
STMY3
LAMC2
S100A4
KLK6
TGFBI
P21
CDC42BPA
OPN__OSTEOPONTIN
PAI1
GADD45B
EGR1
IGFBP3
IGFBP7
TIMP3
LOXL2
SFRP4
PDGFC
TGFB3
SFRP2
INHBA
CTHRC1
FAP
SPARC
COL1A1
BGN
TIMP2
ANTXR1
ITGB1
DLC1
CALD1
AKT3
AKAP12
Average Distance Between Clusters
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Stage II
Stage III
Stromal Genes
Cell CycleGenes

19. Similar Coexpression of Recurrence Score Genes Stage II vs Stage III
Stage II Development Studies (n=634)
Stage III Development Studies (n=844)
BGN
BGN
INHBA
FAP
FAP
INHBA
GADD45B
GADD45B
cMYC
cMYC
MYBL2
MYBL2
Ki_67
Ki_67
0.00
0.25
0.50
0.75
1.00
1.25
0.00
0.25
0.50
0.75
1.00
1.25
Average Distance Between Clusters
Average Distance Between Clusters
No evidence of interaction between RS and Stage (p=0.87)

20. Stage II Development Studies (n=634)
Coexpression of Recurrence Score Genes Consistency in Stage II Across Development Studies and QUASAR
Stage II Development Studies (n=634)
Stage II QUASAR (n=506)
BGN
BGN
INHBA
INHBA
FAP
FAP
GADD45B
GADD45B
cMYC
cMYC
MYBL2
MYBL2
Ki_67
Ki_67
0.00
0.25
0.50
0.75
1.00
1.25
0.00
0.25
0.50
0.75
1.00
1.25
Average Distance Between Clusters
Average Distance Between Clusters

21. Comparison with PETACC-3 Molecular Study
Results consistent with PETACC-3
PETACC-3: only MMR and 18qLOH had significant interaction with stage
MMR/MSI appears to be a stronger prognostic marker in stage II than in stage III in both studies
Important differences with PETACC-3
Quantitative RT-PCR
Number of molecular markers assessed
Requirement for ≥12 nodes examined for stage II
All PETACC-3 patients received chemotherapy

22. Summary and Conclusions
Stage II and stage III colon cancer are remarkably similar for expression of the 375 cancer-related genes tested
Cannot rule out stage-specific differences in other molecular markers that were not assessed
For markers which differ between Stage II and Stage III, including MMR and a small number of genes, additional study is warranted to examine clinical relevance
The 12-gene Recurrence Score, validated in Stage II colon cancer, appears to be stage-independent and should be tested in Stage III colon cancer