Advances in Colorectal
Cancer Biomarker
Discovery
Stan Hamilton, MD
Head, Pathology and Laboratory Medicine

• Risk assessment
• Exposure Assessment
• Screening
• Surveillance
• Diagnosis
• Prognosis
• Prediction
• Monitoring

• Exploratory (correlative) studies using clinically annotated biospecimens and research assays
• “Retrospective-prospective” studies using clinically annotated biospecimens, known clinical outcomes, and analytically validated assays
Modified from Dr. Richard Schilsky

• Prospective biomarker/drug co-development studies
• Prospective biomarker development studies
• Prospective biomarker validation studies
Modified from Dr. Richard Schilsky

• Prognostic and predictive markers
• Assays for markers and targets
• Subpopulation, niche, enrichment trials
• Rare tumors
• Discovery, test, validation studies
• Both level of evidence (Level I from prospective marker-directed trials) and weight of evidence

• Integral studies : Tests must be performed in order for the trial to proceed, i.e. tests are essential to the trial (includes markerdirected trials with CLIA compliance).
Modified from Dr. Richard Schilsky

• Diverse solid-tumor types
• Logistics for tumor tissue and control specimens
• Biomarker assay resources
• Regulatory compliance with Clinical
Laboratory Improvement
Amendments of 1988 (CLIA-88)
• Turnaround time for risk assessment and therapy assignment
7

• Integrated studies : S tudies that are intended to identify and validate assays or marker tests that might be used in future trials, but the assay results are not used to make decisions in the current trial.
Modified from Dr. Richard Schilsky

• Correlative studies : Studies to develop biomarkers/assays or imaging tests that are performed retrospectively, are exploratory in nature, and do not meet the criteria of being an integral or integrated study
Modified from Dr. Richard Schilsky

• Define intended clinical use
• Prospectively study the population and specimens for the intended use
• Use an analytically validated biomarker test
• Hypothesis and sample size must be adequate to demonstrate improved clinical outcomes when the biomarker test is applied.
From Dr. Richard Schilsky

• If we are confident that the therapy will not work in marker-negative patients
AND
• We have a validated assay that can reliably assess the status of the marker
THEN
• We might design and conduct clinical trials only in marker-positive patients
Modified from Dr. Richard Schilsky

Marker+
Randomization
Marker−
Randomization
Targeted
Therapy
Standard
Therapy
Targeted
Therapy
Standard
Therapy

High-Risk Patients
18q LOH are
RANDOMIZED
Stratify:
Disease stage
IIA or IIB
Microsatellite instability
(stable/low vs high)
18q LOH
Low-Risk Patients
MSS/MSI-L with retention of 18q alleles or MSI-H are OBSERVED
MSI-L = low-level microsatellite instability
MSI-H = high-level microsatellite instability
*Bevacizumab continued for an additional 6 months
Arm A: mFOLFOX6 q2w × 12
Arm B: mFOLFOX6 + bevacizumab* q2w × 12
Arm C:
Observation only

TAILORx
NODE NEGATIVE BREAST CANCER STUDY
ER/PR + tumors
ONCOTYPE DX ASSAY
Score < 11
29% of pts
Endocrine
Therapy
Score 11-25
44% of pts
R
Endocrine
+
Chemotherapy
Score >25
27% of pts
Chemotherapy +
Endocrine Therapy
Accrual goal= 4800 randomized patients, 11000 screened
Non inferiority = decrease in 5 year DFS from 90 to 87% or less
(Slide courtesy of Dr. Richard Schilsky)

• Adequacy of biospecimen acquisition, processing and storage
• Access to CLIA-certified labs
• Funding for biomarker studies
• Regulatory requirements
• Contractual agreements with commercial partners
Modified from Dr. Richard Schilsky

Advantages of Centralized Core Labs
• Standardization for trials
– Sample collection, processing, and assays
• Expertise of trained personnel
• Availability of state-of-the-art technologies
19

Advantages of Centralized Core Labs
• Assay development, validation, consultation, and interpretation
• Quality and reliability
• Cost-effectiveness
• Uniform access to non-renewable specimens for investigators
20

Integral Biomarker Specimen Flow – E5202
Fax results – avg 4 working days
5 working days d a y s
6
0 m a x
Surgery
Site registers patient to
Treatment
Site registers patient, ships
2 blocks
(1 tumor,
1 normal)
ECOG
Rando
PCO-RL*
Laboratory
QC and
Processing
MDACC* tests for 18qLOH,
MSI
Fax results – avg 4 working days
*CAP-certified lab for CLIA-88 compliance
21

Advantages of Decentralized Labs
• “Real-world”
• Access
• Convenience
22

Biomarkers in CRC:
Recent Advances
• Complexity of microRNA alterations in the adenoma-adenocarcinoma sequence
• Gene expression profiling for prognosis in Stage II colon cancer
• Markers for EGFR antibody therapy
• Heterogeneity
23

He L. et al. (2004) Nat Rev Genet: 522–531
• miRNA
– Cell differentiation
– Cell cycle progression
– Apoptosis
– Regulation of gene expression
• Over 100 miRNAs implicated in colorectal cancer

Mucosa-Adenoma-Adenocarcinoma Sequence
NNM ALG AHG CA

p < 0.001

Red: p< 0.01
Grey: p< 0.05
Black: p> 0.05
Significant Pairwise Comparisons for 230 miRS hsa-miR-224 hsa-miR-877* hsa-miR-1 hsa-miR-632 hsa-miR-130a
NM: Non-neoplastic mucosa
ALG: Adenoma with lowgrade dysplasia
AHG: Adenoma with highgrade dysplasia
CA: Adenocarcinoma

Example of Group 1A:
Early Persistent
Example of Group 2B:
Late

• Large number of miRNAs deregulated in progression from non-neoplastic mucosa to adenoma to adenocarcinoma
• Complex patterns of dysregulation dependent on the phase in progression
• Dysregulation often an early event
• Use of miRNAs as biomarkers or as therapeutic targets or agents dependent upon the timing of altered expression.

Biomarkers in CRC:
Recent Advances
• Complexity of microRNA alterations in the adenoma-adenocarcinoma sequence
• Gene expression profiling for prognosis in Stage II colon cancer
• Markers for EGFR antibody therapy
• Heterogeneity
30

The 12-Gene Oncotype DX ®
Score ®
Colon Cancer Recurrence
7 CANCER RELATED GENES
Cell Cycle Stromal
Ki-67
C-MYC
MYBL2
FAP
BGN
INHBA
GADD45B
5 R EFERENCE G ENES
ATP5E PGK1 GPX1 UBB VDAC2

QUASAR Results: Colon Cancer Recurrence
Score ® Predicts Recurrence Following Surgery
Prospectively-Defined Primary Analysis in Stage II Colon Cancer (n=711)
35%
30%
25%
20%
15%
10%
5% p=0.004
0%
| | ||||| | | | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| || ||| ||||||||||| | || | |||||| |
0 10 60 20 30 40
Recurrence Score
50 70
Kerr et al., ASCO 2009, #4000

QUASAR Results: Recurrence Risk in
Pre-specified Recurrence Risk Groups
1.0
Recurrence
Risk Group
Low
Range of RS
Proportion of patients
<30 43.7%
Intermediate 30-40
High ≥41
30.7%
25.6%
0.8
0.6
Comparison of High vs. Low
Recurrence Risk Groups using Cox
Model: HR = 1.47
(p=0.046)
0.4
0.2
0.0
0 n=711
Recurrence Risk Group
Kaplan-Meier Estimates (95% CI) of Recurrence Risk at 3 years
Low
Intermediate
High
12% ( 9% -16%)
18% (13%-24%)
22% (16%-29%)
1 2 3
Years
4
Kerr et al., ASCO 2009, #4000
5

Biomarkers in CRC:
Recent Advances
• Complexity of microRNA alterations in the adenoma-adenocarcinoma sequence
• Gene expression profiling for prognosis in Stage II colon cancer
• Markers for EGFR antibody therapy
• Heterogeneity
34

JNCI 101:1310, 2009

PLoS One 4: e7287, 2009

PLoS ONE 4: e7287, 2009

Biomarkers in CRC:
Recent Advances
• Complexity of microRNA alterations in the adenoma-adenocarcinoma sequence
• Gene expression profiling for prognosis in Stage II colon cancer
• Markers for EGFR antibody therapy
• Heterogeneity
38

9

PROJECT T 9 Two-stage analysis
Sequenom screen Orthologous confirmation
Dynamic Sanger Sequencing
Any activating mutations in >5% in any major tumor lineage
PI3K/AKT
Pathway
MEK
Pathway
Receptors
AKT1, 2, 3
PIK3CA
PHLPP2
FRAP (mTOR)
RICTOR
PDPK1
PIK3R1
BRAF
HRAS
KRAS
MEK1,2
NRAS
RAF1
PRKAG1/2
MC1R
EGFR
FGFR1,2,3
KIT
VEGF
PDGFRA
GNAQ
ERa
MET
ALK
ABCB1
Downstream
Effectors
CDK4
CTNNB1
FBXW7
JAK2
RET
FLT3
IDH1,2
Dear1
TNK2(ACK1)

Heterogeneity of Biomarkers
• Intra-tumoral
• Primary cf. synchronous metastasis
• Multiple metastases
• Primary cf. metachronous recurrence
• Recurrence cf. recurrence after chemotherapy
41

Heterogeneity of Biomarkers
• Co-mutation heterogeneity:
The rule, not the exception
• Discordance varies with genes
• Primary cf. synchronous liver metastasis
– KRAS: 30%, most acquired
– NRAS: 100%, 75% acquired and
25% lost
42

• Ability to complete the various types of biomarker studies including validation trials to contribute to personalized cancer care
• Large numbers of patients required

• Funding
- Patient accrual (cf. industry trials)
- Effort of faculty for salary support
- Marker studies
Sources
Phasing with protocol development

• Complexity and duration of protocol review process
• Regulatory issues
• Informatics
• Markers to be valued and addressed like drugs
• “A bad marker is as harmful as a bad drug.”