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André Tartar [email protected]
The Traditional Treatment Paradigm
Trial and Error
Symptoms
Diagnosis
Treatment
Dosing
Nonspecific
Broad
Nonselective
One-size-fits-all
Phenotype
Evidence-Based Medicine
Biomarkers can be:
Anatomical
 Histological
 Imaging
 Genes
 mRNA
 Proteins
 Metabolites
Essentially, some aspect of biology that has predictive power
Biomarkers can be used for:
 Diagnosis
 Presence of disease or identify at-risk patients
 Staging of disease (severity)
 Prognosis of disease (metastases)
 Stratification of patients
 Responders/non-responders
 Monitoring/predicting Toxicity
 Monitoring therapeutic or pharmacodynamic
effect
Letting the Genome out of the Bottle
Will We Get Our Wish?
NEJM
A patient, perhaps one you have known for years,
who is overweight and does not exercise regularly,
shows up in your office with an analysis of his
whole genome at multiple single-nucleotide
polymorphisms (SNPs).
His children, who were concerned about his
health, spent $1,000 to give him the analysis as a
holiday gift.
N Engl J Med 2008; 358:105-107January 10, 2008
ILLUMINA JANUARY 2014
http://www.illumina.com/technology/next-generation-sequencing.ilmn
The ultimate goal of pharmacogenomics
is delivering the right dose of the right
medicine to the right patient
Genomic and PGx
Inter-individual variations
Efficacy and adverse reactions
Environmental factors
Climat, Diet, Disease
organisms or toxic
substances, Lifestyle factors,
Weight, Age, Sex, Other
medicines
Genetic factors
3 billions nucleotides
Inter-individual
Variations ~ 3
millions
85% of patient responses to a drug are linked to genetic
factors.
DNA based tests: increase efficacy and safety;
pharmacogenomics (pharmacology + genomics)
Genetic Factors influence drug responses
via 3 routes:
By affecting
the processing
of the drug by
the body
By modifying
the proteins
that are
targeted by the
drug
By influencing
the risk of
acquiring
certain
diseases
PHARMACO
-KINETICS
PHARMACO
DYNAMICS
DISEASE
PATHWAYS
More than 120 FDA-approved drugs have pharmacogenomics
information in their labeling (Dec. 2013)
By affecting
the processing
of the drug by
the body
1/ PGx of
Metabolic
Enzymes
Clinical PGx
PK factors determining an individual’s drug reaction
Hyper
metabolisers
Drug cleared
too quickly
NO EFFECT
Extended
metabolisers
DESIRED
EFFECT
Poor
metabolisers
Drug slow to be cleared from the body
RISK OF ADVERSE DRUG
REACTION
Evolution of Clinical Pharmacogenomics
Phenotype
Individual
genes
GWAS
Sequencing
Probe drug
PCR-based
Increasing use
Future?
Metabolite:
parent drug
ratio
Current
Standard
Robust findings
Implementation
Thiopurine Metabolism
Phenotypic analysis
Thiopurine Metabolism
Active metabolite
Important
enzyme
CH3
CH3
Inactive metabolites
15
Azathioprine used to treat neoplasias is converted to 6-mercaptopurine in vivo.
6-mercaptopurine
Azathioprine
20 years ago it was reported that patients can be separated into three groups on
the basis of the level of TPMT activity in their red cells.
When patients with low levels of TPMT activity received standard doses of
thiopurines, they had a greatly increased risk of life-threatening, drug-induced
myelosuppression.
Level of TPMT activity in red cells among 298 randomly selected white adult blood donors
In the case of TPMT, a blood sample could be obtained and the enzymatic activity measured directly,
The fact that TPMT is expressed in an easily accessible cell facilitated the introduction of this test.
If for instance an hepatic enzyme like CYP2D6 would have been involved a probe drug had to be
administered and a urine sample collected.
Evolution of Clinical Pharmacogenomics
Phenotype
Individual
genes
GWAS
Sequencing
Probe drug
PCR-based
Increasing use
Future?
Metabolite:
parent drug
ratio
Current
Standard
Robust findings
Implementation
Thiopurine Metabolism
Genotypic Analysis
From TPMT
Phenotype to
Genotype
The human TPMT gene: TPMT*1 is the most common allele, and TPMT*3A
is the most common variant allele among white subjects
When mercaptopurine (MP) treatment is given at conventional doses
- TPMT-deficient patients (v/v pink) show a markedly (tenfold) higher systemic
exposure to active thioguanine nucleotides (TGN)
- Heterozygous patients (wt/v blue) show about twofold higher TGN concentrations.
The higher concentrations of TGN translate into a significantly higher frequency of
haematopoietic toxicity
Cytochrome P450
Genotypic Analysis
Drug Metabolizing Enzymes
The liver is the major site of drug metabolism.
The AmpliChip tests are based on
Affymetrix microarray technology
* labeled DNA target
*
* *
*
*
Oligonucleotide probe
CYP450 2D6 & 2C19
To address the relevant
genetic variations, each array
contains over 15,000 different
probes complementary to
sense and anti-sense P450
genomic DNA.
Probes range in length from
18mer to 22mer
Cytochrome P450 2D6
Genotypic Analysis
Genotype-Phenotype Relationship of
CYP2D6 polymorphism
Genotype
or
or
or
Phenotype
PM
IM
EM
URM
Frequency
(5-10%)
(10-15%)
(65-80%)
(5-10%)
# of individuals
(Caucasians)
Increasing enzyme activity
31
Cytochrome P450 2C19
Clopidogrel
Clopidogrel (prodrug)
Active metabolite
n engl j med 360;4 nejm.org january 22, 2009
162 healthy subjects receiving clopidogrel
- Pharmacokinetic: Plasma concentrations of the active metabolite of clopidogrel
were measured by HPLC-MS.
- Pharmacodynamic : absolute reduction in maximal platelet aggregation from
baseline in response to 20 μM of ADP.
n engl j med 360;4 nejm.org january 22, 2009
Method: We tested the association between functional genetic variants in CYP
genes, plasma concentrations of active drug metabolite, and platelet inhibition in
response to clopidogrel in 162 healthy subjects.
Results: In healthy subjects who were treated with clopidogrel, carriers of at least
one CYP2C19 reduced-function allele (approximately 30% of the study population)
had a relative reduction of 32.4% in plasma exposure to the active metabolite of
clopidogrel, as compared with noncarriers (P<0.001).
Experimental: Genomic DNA was extracted from whole-blood.
Genotyping was performed after amplification by means of a PCR assay involving
two primers for the major variant alleles CYP2C19*2 , CYP2C19*3 and
CYP2C19*4, CYP2C19*5, CYP2C19*17.
Effects of at least one reduced-function allele in five genes encoding cytochrome
P-450 on the pharmacokinetic and pharmacodynamic responses to clopidogrel
Primary efficacy outcome: death from cardiovascular causes, myocardial infarction, or stroke
Background
Pharmacogenetic determinants of the response of patients to clopidogrel contribute to variability in the
biologic antiplatelet activity of the drug. The effect of these determinants on clinical outcomes after an acute
myocardial infarction is unknown.
Methods
2208 patients presenting with an acute myocardial infarction receiving clopidogrel therapy.
Relation of allelic variants of genes modulating clopidogrel absorption (ABCB1), metabolic activation
(CYP3A5 and CYP2C19), and biologic activity (P2RY12 and ITGB3) to the risk of death from any cause,
nonfatal stroke, or myocardial infarction
Estimated Rates of Death from Any Cause, Nonfatal Myocardial Infarction, or
Stroke, according to the number of CYP2C19 loss-of-function alleles.
Initiation of antiplatelet therapy based on cytochrome P450 2C19 genotype in
patients with coronary disease following percutaneous coronary intervention
Evolution of Clinical Pharmacogenomics
Phenotype
Individual
genes
GWAS
Sequencing
Probe drug
PCR-based
Increasing use
Future?
Metabolite:
parent drug
ratio
Current
Standard
Robust findings
Implementation
SNPs Mutations in the Genome
• One in every 1200 bases
may be different in any
two humans.
• This variation, which is
called a polymorphism, is
largely responsible for
differences between how
humans respond to drugs.
Several types of mutations
are associated with these
variations.
Mutations in the Genome
In addition to modifying the function expressed protein, mutations can also affect
the level of gene expression when they occur in regulatory or promoter sequences
or in the exon/intron boundary.
A genome-wide association study (GWAS), is an examination of many common
SNPs in different individuals to see if any variant is associated with a trait.
These studies normally compare the DNA of two groups of participants: people with
the disease (cases) and similar people without (controls).
In contrast to methods which specifically test one or a few genetic regions, the
GWA studies investigate the entire genome.
A Manhattan plot depicting strongly associated risk loci. Each dot represents a SNP,
with the X-axis showing genomic location and Y-axis showing association level.
Price: $199.00
The Illumina Omni Express array is
a 12-sample BeadChip microarray.
It includes a total of 730,525
genome-wide markers and targets a
minor allele frequency of 5%
The EMA decision-making tree for in vitro studies prior to human exposure and Phase I studies
The EMA decision-making tree for in vitro studies prior to human exposure and Phase I studies
The European Medicine Agency’s decision-making tree for Phase I and Phase II studies.
Evolution of Clinical Pharmacogenomics
Phenotype
Individual
genes
GWAS
Sequencing
Probe drug
PCR-based
Increasing use
Future?
Metabolite:
parent drug
ratio
Current
Standard
Robust findings
Implementation
May 09 2011
NEW YORK (GenomeWeb News) –
Illumina today said that it has lowered the cost of its human
whole-genome sequencing services to:
$5,000 per genome for projects of 10 samples or more,
$4,000 for projects of 50 samples or more.
19 Novembre 2013:
http://www.nejm.org/doi/full/10.1056/NEJMp1314561?query=featured_home
ILLUMINA JANUARY 2014
http://www.illumina.com/technology/next-generation-sequencing.ilmn
By influencing
the risk of
acquiring/
developping
certain
diseases
2/ Patients and
Pharmacogenomics
Patients and PGx: Biomarkers
Healthy
Asymptomatic
disease
Risk
Assessment
Screening/
Diagnosis
Prognostic
Predictive
Monitoring
Predispositio
n for
developing
disease
Early
detection
Predict
probable
disease course
Predict likely
response to a
drug
Monitor
treatment
efficacy/ disease
recurrence
Symptomatic disease
Chronic disease/
Cured
Patient Stratification / Therapy Selection
Therapy adaptation
Breast Cancer
Genotypic Analysis of recurrence risk
Oncotype DX is a 21-gene assay that:
 quantifies the likelihood of disease recurrence in early-stage breast cancer
 assesses the likely benefit from certain types of chemotherapy.
Benefit of Chemotherapy Depends on Recurrence Score
Absolute Increase in DRFS at 10 Years
n = 353
Low
RS<18
n = 134
Int
RS18-30
n = 164
High
RS≥31
0
10%
20%
30%
40%
% Increase in DRFS at 10 Yrs (mean ± SE)
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3227972/pdf/onc457.pdf
Guidelines for hormone receptor (HR)–positive early breast cancer recommend
addition of adjuvant chemotherapy for most women, leading to overtreatment,
which causes considerable morbidity and cost.
The cost-effectiveness of recurrence score (RS)–guided treatment using 21-gene
assay (Oncotype DX) was compared with treatment guided by the Adjuvant!
Online program (AOL).
A validated software program Adjuvant! Online (AOL), projects outcomes at 10
years based on clinico-pathological features and therapy.
It is freely available on the Internet and is now widely used to assist in the adjuvant
treatment decision-making process.
Cost of the 21-gene assay, Oncotype DX, was based on the manufacturer’s
suggested retail price of 4102 CAN $ ($3,650 USD).
1
All patients start in the risk classification state where they undergo risk assessment
by AOL with or without RS reclassification
Treatment strategies are also determined at this state.
1- Patients receive chemotherapy if they are considered high risk by RS
reclassification (strategy 1) or high risk by AOL assessment only (strategy 2).
Patients enter the chemotherapy state for the next 6 months.
2
They may experience no, minor, or major toxicity or may die from complications
related to chemotherapy.
2- After 6 months of chemotherapy, they transit to the recurrence- free state.
3
3- Patients who are deemed low risk by RS reclassification (strategy 1) or low risk
by AOL assessment only (strategy 2) proceed directly to the recurrence-free state
and complete 5 years of tamoxifen
4
During each cycle, these patients may remain disease-free or develop distant
recurrence.
4- Once patients develop distant recurrence, they remain in the distant recurrence
state until they die from breast cancer.
3/ Drug Discovery
By modifying
the proteins
that are
targeted by the
drug
PHARMACO
DYNAMICS
and Development
and
Pharmacogenomics
Pharmacogenomics of drug target
Co-developement of NCE and Test
Herceptin:
Co-developed
with Herceptest
HER2-positive testing (a) by IHC and (b) by FISH.
FFPE:
formalin-fixed
paraffinembedded
KRAS testing for Erbitux in
colorectal cancer
Retrospective Analysis
Erbitux and K-ras
ERBITUX binding blocks EGFR
signal transduction and inhibits
downstream intracellular signalling
Mutation of the K-ras gene results in
a constitutively activated K-ras
protein, which signals WITHOUT
prior EGFR-mediated signalling
The anti-tumor effects mediated by
EGFR blockade are bypassed by
mutated K-ras
82
Erbitux, retrospective Analysis:
572 patients with colorectal cancer
were randomly assigned to receive
cetuximab plus best supportive care or
best supportive care alone.
Tumor samples, obtained from 394 of
572 patients were analyzed to look for
activating mutations in exon 2 of the Kras gene.
Improved survival with cetuximab was
only observed in non mutated (wild
type) K-ras group.
Erbitux
Prognostic vs. Predictive
Markers
Prognostic
marker
Predictive
marker
Influences clinical
outcomes
independent of
therapy used
Influences clinical
outcomes with a
specific therapy
Markers may correlate with prognosis and treatment outcome
ER, HER2 in breast carcinoma
KRAS, BRAF in colorectal carcinoma
MGMT in glioblastoma
Conclusions
Industry perspectives on personalized medicine
http://www.nature.com/nrd/journal/v11/n3/pdf/nrd3677.pdf
Percentage of company (21) pipeline relying on biomarker data
Fewer projects (10% or less) had identified specific target populations or
companion diagnostics
Nature Reviews Drug Discovery 11, 178 (March 2012)
Number of times respondents mentioned personalized
medicines for particular therapeutic areas
Nature Reviews Drug Discovery 11, 178 (March 2012)
Scientific potential and economic attractiveness
for companion diagnostics development across therapeutic areas.
Erbitux and KRAS
94
•
ERBITUX binding
blocks EGFR signal
transduction and
inhibits downstream
intracellular signaling
•
Mutation of the KRAS
gene results in a
constitutively activated
KRAS protein, which
signals WITHOUT prior
EGFR-mediated
signaling
•
The anti-tumor effects
mediated by EGFR
blockade are bypassed
by mutated KRAS