Predictive Biomarkers

Ior tailoring drug therapy

utline
1. What is a predictive biomarker and why are they
important?
2. How does genomics Iacilitates the search Ior
predictive biomarkers?
3. Are there any examples oI clinically useIul
predictive biomarkers?
1. What is a predictive
biomarker?
Predictive Biomarkers
Biomarker: A gene, protein, or other change that
heralds a biomedical phenotype before that
phenotype s clinically apparent.
Biomarkers used Ior: Diagnosing Disease
Establishing Disease Risk
Establishing Drug Response
Establishing Drug Toxicity
Predicting Drug Response
Predicting Drug Toxicity
hat is the most valuable
Predicitive Biomarker
today?
D
High D (low density lipoprotein) levels indicate individuals
At high risk oI artherosclerotic plaque Iormation.
Artherosclerotic plaque Iormation leads to thrombosis (stroke) and
Embolism (heart attack)
Stroke and heart attach are the major killers in the world
The use oI D as a biomarker oI heart disease has led to the
explosion oI the statins: multi-billion cholesterol reducing drugs.
unctional Genomics: Genome-wide biomarker drug discovery
2. How genomics is
Iacilitating the search Ior
predictive biomarkers?
&sing the sequence inIormation to probe the transcriptome oI the Cell
Transcriptome: the set oI all expressed genes (mRNAs) inside a cell.
DiIIerent types oI technology used to probe the transcriptome:
SAGE (serial analysis oI gene expression)
Strategy: isolated mRNA is reversed-transcribed to cDNA, digested into Iragments,
randomly ligated into clones, and the clones are sequenced. Relative abundance oI
diIIerent mRNAs within a single pool are reIlected in the sequenced clones.
DNA microarrays
Strategy: mRNA is reversed transcribed to cDNA, labeled with Iluorescent probe,
And hybridized to complementary strand DNA attached to glass slides. By
comparing hybridizationn oI two samples, relative abundance oI mRNA between samples
can be determined
AIIymetrix GeneChips. Same as DNA microarrays, except that cDNA is transcribed to
cRNA in vitro, which is labeled with Iluorescent probes.oligonucleotides are chemically
synthesized on chip and the presence/absence oI genes is determined based on
hybridization between sets oI perIectly matched and mismatched probes.
SAGE: Serial Analysis oI Gene Expression
AAAA
AAAA
AAAA
AAAA
AAAA
AAAA
Cut and tag
Concatenate
Sequence
ATTGCGTACCCCgtacTTGAG
AACCCgtacATTGCGTACCCC
gtacGCCCAATTCATTTAgtacA
TTGCGTACCCCgtacAAAACC
CCTTTTT
gtac
gtac
gtac
gtac
gtac
gtac
Total cell mRNA
A B C D E
5 3 12 4 1 12
DiIIerential Analysis oI Gene Expression with SAGE
A B C D E
4 2 10 5 1 4
Normal Sample Disease Sample
Gene
# SAGETags

NormaI SampIe
D
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a
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e

S
a
m
p
I
e
x
1SD
2SD
F|uorescerl prooes prepared lror lWo F|uorescerl prooes prepared lror lWo
rRNA sources lo oe corpared rRNA sources lo oe corpared
Cy Cy--dCTP ruc|eol|des added lo lre c0NA dCTP ruc|eol|des added lo lre c0NA
syrlres|s reacl|or syrlres|s reacl|or
Cy3 (greer) Cy3 (greer)--corlro| sarp|e corlro| sarp|e
Cy5 (red) Cy5 (red)--exper|rerla| sarp|e exper|rerla| sarp|e
Probe Preparation and labeling Ior hybridization experiments
Synthetic oligonucleotide arrays: AIIymetrix Gene Chips
PM
MM
DiIIerential Analysis oI Gene Expression with GeneChip
A B C D E
4 2 10 5 1 4
Normal Sample (Red) Disease Sample(Green)
Gene

NormaI SampIe
D
i
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a
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S
a
m
p
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x
1SD
2SD
PM-MM
5 3 12 4 1 12
A B C D E
||goruc|eol|des are oola|red oy PCR ||goruc|eol|des are oola|red oy PCR
arp||l|cal|or ol c0NA lerp|ales ard rave ar arp||l|cal|or ol c0NA lerp|ales ard rave ar
ar|re ||r||rg group or 5' erd ar|re ||r||rg group or 5' erd
0|ass s||des are crer|ca||y lrealed (a|deryde) 0|ass s||des are crer|ca||y lrealed (a|deryde)
lo oe reacl|ve lo lre group or lre 5' ol lre o||go lo oe reacl|ve lo lre group or lre 5' ol lre o||go
A r|crodrop|el ol PCR producl |s p|aced or A r|crodrop|el ol PCR producl |s p|aced or
s||de W|lr use ol rela| p|r. 3cr|ll oase reacl|or s||de W|lr use ol rela| p|r. 3cr|ll oase reacl|or
||r|s o||go lo s||de surlace v|a cova|erl oord. ||r|s o||go lo s||de surlace v|a cova|erl oord.
Spotted arrays: cDNA microarrays
DiIIerential Analysis oI Gene Expression with Microarrays
A B C D E
4 2 10 5 1 4
A B C D E
5 3 12 4 1 12
Normal Sample (Red) Disease Sample(Green)
Gene
luorescence

NormaI SampIe
D
i
s
e
a
s
e

S
a
m
p
I
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x
1SD
2SD
How transcriptome analysis oIIers insights into potential biomarkers
DiIIerential gene expression in normal tissues
(relating the expression oI speciIic genes to suceptibility
I certain tissue to speciIic diseases (e.g. retinal degeneration).
DiIIerential gene expression in disease states.
(comparing normal and disease tissues; class prediction; class
IdentiIication.)
Gene expression in model systems
(comparing drugs to mutations, human to mice, cells to
iving tissues)
Gene expression in pathogens
Gene expression in response to drugs
(drug target identiIication, drug classiIication; drug activity,
Drug metabolism)
Proteomics inding Protein Biomarkers
30,000 genes
~200,000 proteins
The Basic Technique: Sodium dodecyl sulIate- polyacrylamide
Gel electrophoresis (SDS-PAGE)
Basic Technique 2: Isoelectric Iocusing
Isoelectric Iocusing
SDS
2D gel electrophoresis: Isoelectric Iocusing x SDS PAGE
PAGE
Comparative analysis oI 2D gels Irom diIIerent samples
Mass Spec sequencing: ID proteins according to Iragment masses
1022-875 147
292-145 147
3. How are predictive
biomarkers making their
way into the clinic?
BIMARKERS &SED IN CANCER THERAP
The two-hit hypothesis: Cancer cells need to acquire both gain-oI-
Iunction ('oncogenic) and loss-oI-Iunction ('tumor suppressor)
Mutations to become cancerous
ncogene:gain oI Iunction
Tumor suppressor: loss
I Iunction
ncogenic mutation mechanisms
Deletion or point mutation
in coding sequence
DNA
DNA
mRNA
protein
Gene ampliIication
Chromosomal
rearrangement
#!%N
Herceptin (Trastuzumab) is the first targeted, humanized
antibody for treatment of women with HER (human epidermal
growth factor receptor ) positive metastatic breast cancer.
Herceptin is designed to target and block the function of HER.
HER positive metastatic breast cancer have a more aggressive
disease, greater likelihood of recurrence, poorer prognosis and
approximately half the life expectancy of women with HER
negative breast cancer.
The U.S. Food and Drug Administration (FDA) approved
Herceptin in September in combination with paclitaxel for
treatment of patients with metastatic breast cancer whose tumors
overexpress the HER protein and who have not received
chemotherapy for their metastatic disease.
Herceptin is one of the few therapies, and the only humanized
antibody, that has demonstrated a survival benefit indicated for
metastatic breast cancer
Herceptin- Antibodies block HER2
IinicaI %riaI #esuIts
Data showed that patients receiving Herceptin with chemotherapy survived for a
median of . months compared to .3 months for patients in the Phase trial
receiving chemotherapy alone, a difference of percent.
n addition to survival, when used in combination with chemotherapy, Herceptin
was shown to improve overall response rates from percent in women treated
with chemotherapy alone to percent with Herceptin added -- a percent
increase. The median time to disease progression increased from . months in
the chemotherapy alone group to 7. months in the Herceptin plus
chemotherapy group -- a 3 percent increase.
Additionally, the duration of response of women treated with Herceptin with
chemotherapy was .3 months, compared to . months for those women
receiving chemotherapy alone.
n the trial evaluating Herceptin alone, percent of women who had failed
one or two prior chemotherapy regimens had objective tumor responses with
tumor shrinkage of percent or greater.

W Allogeneic bone marrow transplantation was the only curative

treatment available but is only applicable to a minority oI patients
(15-20).
W Chronic Myelogenous eukemia: a biphasic disease
a. chronic phase (associated with t(9;22)
translocation.)
b. blast crisis (Iatal Ior 85 oI Patients)
.
ther example: Targeted Inhibitors oI BCR-AB
oncogene
W A recent advance in the treatment has been the introduction oI the
Bcr-Abl inhibitor STI571 (Gleevec)
Diagnosis: t(9;22)
ISH: &RESCENCE IN SIT& HBRIDIZATIN
NRMA M&TANT
Karyotype
analysis
STI-571 bound to
AB (PDB: 1IEP)
Rational design:
Ieevec binds Bcr-AbI protein target
Initially, 90 Remission with GEEVEC with
recurrence due to resistance (mediated by BCR-
AB mutations)
Anti BCR-AB cocktails to Iight oII resistant cells.
Ability to target c-kit kinase has led to additional
application: gastric cancer.
Current market value over 1 billion dollars.
Gleevec clinical results
Transcriptome Analysis Ior Personalized Therapy
Collect tumor sample Irom patient
PerIorm transcriptome analysis oI tumor sample
relative to control sample (gene expression proIiles)
Treat tumor
Establish sets oI genes that are up or down-regulated
in responsive vs. non responsive tumors
&se those genes to predict outcome or
Guide treatment decisions
Molecular classiIication oI lung cancers with DNA microarrays
High Throughput Sequencing
Genomic DNA
Cut, polish, add end tags
Attach to beads in emulsion
AmpliIy on bead emulsion
Parallel (multiplexed) sequencing
Assemble reads
Parallel sequencing by
Pyrosequencing
W Roche 454 Pyrosequencing (Add NTPs individually to all the beads, and detect which
bead incorporates the NTP through PPi release. PPi released is added to AMP, to
generate ATP to serve as substrate Ior luciIerase.
A Sequencing Method Based on Real-
Time Pyrophosphate
Mostafa Ronaghi, Mathias Uhln, and Pal
Nyrn*
Science 17 July 1998:
Vol. 281. no. 5375, pp. 363 - 365
Pyrosequencing Result
A Sequencing Method Based on Real-Time Pyrophosphate
Mostafa Ronaghi, Mathias Uhln, and Pal Nyrn*
Science 17 July 1998:
Vol. 281. no. 5375, pp. 363 - 365
Science 17 July 1998: Vol. 281. no. 5375, pp. 363 -
365
Color-coded Reversible Terminator
Sequencing
Four-color DNA sequencing with 3'-O-modified
nucleotide reversible terminators and chemically
cleavable fluorescent dideoxynucleotides.
J. Guo, N. Xu, Z. i, S. Zhang, J. Wu, D. H.
Kim, M. Sano Marma, Q. Meng, H. Cao, X. i,
et al. (2008)
PNAS 105, 9145-9150
Single Molecule Sequencing
Single-Molecule DNA Sequencing of a Viral Genome
Timothy D. Harris, et al. $.03.0 April 2008:
Vol. 320. no. 5872, pp. 106 - 109