Molecular Subtypes:
Ready for Prime Time
Wells Messersmith, MD, FACP
Professor
Director, Gastrointestinal Medical Oncology Program
Co-Head, Division of Medical Oncology
Program co-Leader, Developmental Therapeutics
March 2014
Conflict of Interest:
1. No employment, speaker’s bureaus, stock ownership,
royalties, patents, etc
2. Data Safety Monitoring Board for OncoMed
3. PI or Local PI of clinical trials by Genentech/Roche,
GSK, Pfizer, Millenium, Bayer, Onconova, and
NIH/CTEP.
Molecular Subtypes
Outline/Objectives:
1. Introduction
2. Molecular subtypes already in use
1. KRAS/NRAS (added wrinkle: L v R?)
2. BRAF/MSI (L v R)
3. Risk Stratification in Adjuvant Setting
4. PI3K
Colorectal Diagnostics: What Was
State of Art in 2008?
CEA – recommended for staging, post-operative, monitoring response
DNA ploidy or proliferation assays – not recommended
p53, TS, DPD, TP – insufficient data
Ras – insufficient data
MSI – not recommended
18q- or DCC - not recommended
Copyright 2009 ASCO
FDA approved tests for colorectal cancer (not widely used):
- EGFR Immunohistochemistry (Dakocytomation PharmDx)
- UGT1A1 Invader Assay (irinotecan glucuronidation) - toxicity
- Circulating tumor cells (CellSearch test) - prognosis
Where have we gone wrong with
“predictive” molecular tests in past?
- EGFR staining and EGFR-targeting mAb’s
- Used to be REQUIRED for insurance approval
- Predictive tests for 5-FU efficacy
- Thymidylate Synthase (TS) expression and
polymorphisms
- Methylenetetrahydrofolate reductase (MTHFR)
polymorphisms
- Dihydropyrimidine Dehydrogenase (DPD)
Deficiency (*2A variants)
- Topoisomerase, ercc1 (studies pending), UGT1A1….
Tests for Cytotoxics: Irinotecan
UDP-glucuronosyltransferase (UGT) 1A1
- Irinotecan’s active metabolite, SN-38, is eliminated
via glucuronidation by UDP-glucuronosyltransferase
1A1 (UGT1A1).
- Patients homozygous for *28 allele (7 vs 6 TA repeats)
have 70% reduction in UGT1A1 activity
- According to U.S. FDA, patients homozygous for *28
“should be considered for dose reduction” based on
small retrospective studies (increased diarrhea in some,
neutropenia in others)
- large studies (FOCUS, N9741, PETACC3) have failed
to confirm clinical usefulness
http://www.fda.gov/cdrh/pdf5/k051824.pdf
Molecular Subtypes
Outline/Objectives:
1. Introduction
2. Molecular subtypes already in use
1. KRAS/NRAS (added wrinkle: L v R?)
2. BRAF/MSI (L v R)
3. Risk Stratification in Adjuvant Setting
4. PI3K
Distinct Biology of R v. L CRC
Analysis of PETACC-3 samples (n=2849)
BRAF mut
MSI
KRAS
PIK3CA
Right
EREG expression
18q loss
20q Gain
EGFR gain
HER2 gain
Mucinous
differentiation
High mutation
Frequency
Poor
Prognosis
Left
Missiaglia, ASCO 2013
Sensitive to
Cetuximab
Good
Prognosis
N0147 Trial: Testing Adjuvant Cetux
Note: in analysis, segregated
“proximal” versus “distal” with
splenic flexure as cut-off
Stage 3
Colon
Cancer
(N = 3768)
Alberts, JAMA 2012
P
R
E
R
E
G
I
S
T
E
R
K-ras WT
Centralized
K-ras
analysis
K-ras
Mut
R
A
N
D
O
M
I
Z
E
R
E
G
I
S
T
E
R
Arm A
mFOLFOX6
Arm D
mFOLFOX6 +
Cetuximab
Arm G
•Adjuvant therapy per
primary oncologist
•Report therapy given
•Annual status through
year 8
Negative Adjuvant Trial: N0147
- No difference in DFS with the addition of cetuximab;
detrimental effect for patients >70, even in KRAS WT group.
- However, trial should yield other useful information…
Alberts, JAMA 2012
N0147: KRAS and BRAF worse
WT/WT
KRAS MT (HR=1.45, For stage IIII
p<0.001)
BRAF MT (HR=1.35,
p=0.0147)
Sinicrope, ASCO 2013
CRC, magnitude
of detrimental
effect of KRAS
and BRAF
mutation is lower
than other
analyses.
KRAS: Proximal v. Distal
Proximal
BRAF MT
KRAS MT
Sinicrope, ASCO 2013
Distal
Adverse
impact of
KRAS
mutations
limited to
distal
cancers;
BRAF not
sitedependent
PETACC-3 (5-FU vs. FOLFIRI): Worse OS (and
RFS) for BRAF Mutants: L>R
left
right
- independent of MSI status, left-sided tumors do worse.
Popovic, ASCO 2013
BRAF/MSI Biology: TCGA
BRAF mutation associated with hypermutated (MSI-H) tumors.
The Cancer Genome Atlas Network Nature 487, 330-337 (2012) doi:10.1038/nature11252
- BRAF mutation analyzed in 506 colon cancer patients from
CALGB 89803 (n=75 BRAF mutated patients)
- Worse overall survival in BRAF mutated patients (HR=1.66); mainly
in microsatellite stable (MSS) patients
- Trend to improvement with irinotecan added to 5-FU/LV (HR=0.52)
in BRAF MT patients, albeit low numbers.
Ogino, Clin Can Res 2012
- Due to confounding effect of MSI status in BRAF MT
patients, Ogino proposed this strategy for classification.
Must split, rather than lump, BRAF MT patients
Ogino, Clin Can Res 2012
What about dMMR and FOLFOX?
Although n<100,
data appear
reassuring that
dMMR patients
derive the same
benefit from the
addition of
oxaliplatin
(HR=0.42, p=.06)
Flejou, ASCO 2013
Molecular Subtypes
Outline/Objectives:
1. Introduction
2. Molecular subtypes already in use
1. KRAS/NRAS (added wrinkle: L v R?)
2. BRAF/MSI (L v R)
3. Risk Stratification in Adjuvant Setting
4. PI3K
Risk & Treatment Benefit Markers
for Stage II Colon Cancer
Recurrence Risk
• Bowel obstruction or
perforation
• T-Stage
• # of nodes assessed
• Tumor grade
• Lymphatic/vascular
invasion
• Margin status
Treatment Benefit
• None
* NCCN Clinical Practice Guidelines for Oncology: Colon Cancer v3.2009
ASCO Recommendations on Adjuvant Chemotherapy for Stage II Colon Cancer, JCO, 2004.
Similar Coexpression of 48 Recurrence Risk
Genes Stage II vs Stage III Colon Cancer
Stromal
Genes
AKAP12
AKT3
ITGB1
ANTXR1
TIMP2
BGN
COL1A1
SPARC
LOXL2
CTHRC1
FAP
INHBA
SFRP2
TGFB3
TIMP3
CALD1
IGFBP7
PDGFC
SFRP4
IGFBP3
DLC1
STMY3
EGR1
GADD45B
PAI1
AKAP12
AKT3
CALD1
DLC1
ITGB1
ANTXR1
TIMP2
BGN
COL1A1
SPARC
FAP
CTHRC1
INHBA
SFRP2
TGFB3
PDGFC
SFRP4
LOXL2
TIMP3
IGFBP7
IGFBP3
EGR1
GADD45B
PAI1
Stage II
OPN__OSTEOPONTIN
CDC42BPA
P21
TGFBI
KLK6
S100A4
LAMC2
STMY3
HSPA1A
GRB10
P14ARF
P16_INK4
CDC20
KI_67
RRM2
MCM2
RRM1
SKP2
CMYC
CSEL1
MYBL2
NME1
UMPS
HNRPD
OPN__OSTEOPONTIN
Cell
Cycle
Genes
Stage III
S100A4
HSPA1A
CDC42BPA
P21
LAMC2
TGFBI
GRB10
KLK6
P14ARF
P16_INK4
CDC20
KI_67
RRM2
MCM2
RRM1
SKP2
HNRPD
NME1
CMYC
CSEL1
MYBL2
UMPS
0.0
0.2
0.4
0.6
0.8
1.0
Average Distance Between Clusters
Benson, ASCO GI Symposium 2014
1.2
0.0
0.2
0.4
0.6
0.8
1.0
Average Distance Between Clusters
1.2
QUASAR RESULTS: Colon Cancer Recurrence
Score Predicts Recurrence Following Surgery
Prospectively-Defined Primary Analysis in Stage II Colon Cancer (n=711)
STROMAL CELL CYCLE
Ki-67
FAP
c-MYC
INHBA
MYBL2
BGN
GADD45B
REFERENCE
ATP5E
GPX1
PGK1
UBB
VDAC2
35%
Risk of recurrence at 3 years
RECURRENCE
SCORE
Group Risk (by Kaplan-Meier)
22%
18%
12%
30%
25%
20%
15%
10%
5%
p=0.004
0%
0
Benson, ASCO GI Symposium 2014
10
20
30
40
50
Recurrence Score
60
70
Recurrence-free interval for (A) all patients (B) stage II,
(C) stage IIIA/B, (D) stage IIIC by Recurrence Score
(RS) groups: NSABP C-07
All patients
II
III A/B
Yothers G et al. JCO 2013;31:4512-4519
IIIC
©2013 by American Society of Clinical Oncology
3 molecular
subtypes of CRC
Subtype A:
“Deficient epithelial”
- Assoc with dMMR
- Epithelial / proliferative
Subtype B:
“Proliferative
Epithelial”
- Low kinome mutations
Subtype C:
“Mesenchymal”
Salazar, ASCO 2013
- Assoc. with EMT
- Worse prognosis
Cancer-Related
Overall Survival
A
Differences
between
curves are
somewhat
modest
Salazar, ASCO 2013
B
C
Subtype C= worse
prognosis
Higher EMT
signature index
in C-type
Salazar, ASCO 2013
No benefit from
adjuvant therapy in
in C-type
Factors in Adjuvant Decision-Making
Sveen A et al. Clin Cancer Res 2013;19:6669-6677
Molecular Subtypes
Outline/Objectives:
1. Introduction
2. Molecular subtypes already in use
1. KRAS/NRAS (added wrinkle: L v R?)
2. BRAF/MSI (L v R)
3. Risk Stratification in Adjuvant Setting
4. PI3K
PI3K
Mutations
(Samuels, Science 2004)
Functionally important:
- Nontruncating
- Nonsynonymous
- Conserved residues
- Higher PI3K activity
©2004 www.sciencemag.org
- Colorectal and gastric cancers frequently harbor mutations.
- Not found in 76 polyps (except two >5cm tubulovillous adenomas)
- Co-existent with KRAS and BRAF mutations (distinct pathway)
How strong is PI3K data?
Retrospective clinical cases; conflicting.
Report
Drugs
Findings
C
7% with PI3K mut’n
Can Res 2006, n=30 (97% chemo) no effect
Lievre
Sartore-Bianchi
Can Res 2009, n=110
Perrone
Ann Oncol 2009, n=32
C and P
13.6% with PI3K mut’n
(67% chemo) lower RR, survival
C
13% with PI3K mut’n
(100% chemo) lower RR, survival
De Roock (#1896) C and P
AACR 2009, n=200
12% with PI3K mut’n
no effect
Di Nicolantonio
AACR 2009, n=132
13% with PI3K mut’n
lower RR, survival
C and P
PI3K as a predictive marker
Prenen, Clinic Can Res 2009, n=200, 12% PIK3CA MT
©2009 American Association for Cancer Research
Exon 9
- 20
- Used sequenome MALDI-TOF
MassArray system
- No relationship between
PIK3CA and KRAS
- No relationship between
PIK3CA and response, survival
Note: PIK3CA mutations have been associated with resistance to trastuzumab in breast
cancer (possible increased dependence on her2/her3 dimerization). Burns, Cancer Cell 2007
Additive Prediction
Response rate increase as markers are added (retrospective database)
DeRoock, Lancet Oncol 2010
PIK3CA mutations and aspirin
Mutant =
aspirin benefit
Liao X et al. N Engl J Med 2012;367:1596-1606
wildtype
Possible Mechanism?
COX2 expression
PGE2
PI3K signal
transduction pathways
regulate COX-2
expression and PGE2
synthesis.
PD = PD098059
(MAPK inhibitor)
Di Popolo et al, Oncogene 2000
LY = LY294002
(PI3K inhibitor)
Conclusions
- The revolution of genetic information on tumors over the
last 5 years has greatly increased understanding of tumor
biology.
- But… this has not yet translated into the clinic, since
most anti-cancer drugs are used empirically.
- Ignorance (lack of research) is costly and deadly; without
knowledge of KRAS, for instance, we would be spending
$700,000,000 per year harming patients in the U.S.
- Knowledge of relevant biomarkers is critical in caring for
colorectal cancer patients; however, important to use
markers that have been validated/qualified across
multiple studies.
Thank You!
wells.messersmith@ucdenver.edu