Dr Howard L McLeod

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Pharmacogenetics

• Dr Howard L McLeod
• Associate Professor of Medicine,
• Genetics, and PharmacologyWashington University,
  St Louis, USA

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A surgeon who uses the wrong side of the scalpel
cuts his own fingers and not the patient if the
same applied to drugs they would have been
investigated very carefully a long time
ago Rudolph Bucheim Beitrage zur
Arzneimittellehre, 1849
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All patients with same diagnosis
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All patients with same diagnosis
Alternate therapy non-responders and toxic
responders
Standard therapy Responders and Patients
Not Predisposed to Toxicity
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• The clinical problem
• Multiple active regimens for the treatment of
  most diseases
• Variation in response to therapy
• Unpredictable toxicity

With choice comes decision
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Many ways to skin a genome
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Biochemical pathways for fluoropyrimidines
MTHFR
MTHF
dFUMP
5-FU
TS
DHF
DPD
dTMP
DNA
F-b-alanine
DPD-dihydropyrimidine dehydrogenase TS-thymidylate
synthase
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Impaired DPD activity from an exon 14 slice site
mutation
Exon 13
Exon 14
Exon 15
at
ag
gt
ag
gt
ag
5
3
Exon 13
Exon 15
5
3
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Biochemical pathways for fluoropyrimidines
MTHF
dFUMP
5-FU
TS
DHF
5
0
0
DPD
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5
0
dTMP
4
0
0
3
5
0
3
0
0
DNA
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5
0
DPD activity (pmol/min/mg protein)
F-b-alanine
2
0
0
1
5
0
1
0
0
5
0
0
DPD-dihydropyrimidine dehydrogenase TS-thymidylate
synthase
wild-type
Proband (/-)
/-
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An anecdote-how interesting!?
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N9741 Schema

• 524 stage IV patients

RANDOMI ZAT ION
IFL Irinotecan 5-FU/LV
FOLFOX Oxaliplatin 5-FU/LV
IROX Irinotecan oxaliplatin
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patients with metastatic colorectal cancer
5FU/CPT-11
5FU/Oxal
CPT-11/Oxal
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5-fluorouracil pathway
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Irinotecan pathway
CPT-11
cell membrane
ABCB1
CPT-11
APC
CYP3A4
CES1
CPT-11
NPC
CYP3A5
CES2
CES1
CES2
SN-38
UGT1A1
SN-38
SN-38G
ABCB1
ABCG2
ABCC1
ABCC2
SN-38
TOP1
ADPRT
XRCC1
TDP1
NFKB1
CDC45L
Cell Death
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UGT1A1 promoter polymorphism and toxicity
UGT1A1 gene structure
Iyer et al 2002
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UGT1A1 TA repeat irinotecan neutropenia
N524
McLeod et al, ASCO 2003
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Association between candidate genotype and
toxicity/efficacy
Key
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We do not know very much about drugs
CPT-11
cell membrane
ABCB1
CPT-11
APC
CYP3A4
CES1
CPT-11
NPC
CYP3A5
CES2
CES1
CES2
SN-38
UGT1A1
SN-38
SN-38G
ABCB1
ABCG2
ABCC1
ABCC2
SN-38
TOP1
ADPRT
XRCC1
TDP1
NFKB1
CDC45L
Cell Death
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Discovery Strategies
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Centre d Etude du Polymorphisme Human (CEPH)
Cell lines

• Large, multigeneration pedigrees widely studied
• Immortalized lymphoblastoid cell lines

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Methodology
Cells counted, plated at 1 x 104 / well
Cells incubated with increasing concentrations of
drug
Alamar blue vital dye indicator added
Viability relative to untreated control
calculated by spectrophotometry
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Advantages of This Model

• Large pedigrees, highly informative for linkage
• Unlimited sample
• Allows for quantitative, accurate phenotyping
• Allows for family studies not possible in humans
• A wealth of genotyping information already
  available
• Likely to address PD component of drug response
• Enables the identification of new candidate genes

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Docetaxel

• Microtubule stabilizing agent used to treat lung,
  breast tumors

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Significant Variation in Cellular Sensitivity to
Docetaxel
427 cell lines analyzed, 38 CEPH reference
pedigrees
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Genome-Wide Linkage Analysis

• Family structure and genotype data allows for
  genome-wide linkage studies
• Carried out using SEGPATH
• Province et al, Genet Epidemiol 24, 128-38 (2003)
• Variance-components method based on IBD sharing
• Collaboration with Aldi Kraja and Mike Province,
  WUSM

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Genetic Markers N983
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Examples of Genes in Chromosome 9 Interval
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Discovery Strategies
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Microarray analysis of extreme samples

• Microarray analysis of docetaxel extremes
• 7 most sensitive, 7 most resistant, 10nM
  docetaxel
• Unrelated
• Affymetrix U133 2 chip

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QTL genes associated with docetaxel sensitivity
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Discovery Strategies
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SNP-Based Association Testing

• Freely available SNP genotype data available
• N3000 Marker source TSC (snp.cshl.org)
• Genotyped in 48-50 full pedigrees
• Ngt1,000,000 Marker source HapMap (hapmap.org)
• Genotyped in 90 individuals (30 trios)
• All SNPs in 1-LOD intervals were tested

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HapMap SNPs in the Chromosome 9 Interval
HapMap
77 M bps
105 M bps
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Discovery Strategies
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Candidate Gene Summary
DAPK1 RHOBTB3
Association
Linkage
C9ORF10 RASSF6 NEK6
MAP1B
Expression
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C9ORF10
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NEK6
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Validation step 1

• Confirmation of candidate genes in validation set
• 100 lymphoblastoid cell lines derived from
  unrelated Caucasian donors (Human Variation
  Panel Caucasian Panel of 100)

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Discovery Strategies
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Inbred Strains as a Discovery Tool in
Pharmacogenomics

• Mouse Phenome Project
• http//aretha.jax.org/pub-cgi/phenome/mpdcgi?rtnd
  ocs/home
• Establish a collection of chemotherapy response
  phenotypes among inbred strains

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• Methods
• 21 different inbred strains
• 5 mice/each strain (all male)
• Baseline neutrophil count assessed by CBC
• 20mg/kg docetaxel injected I.P.
• Neutropenia assessed by CBC at days 1, 3, 4, 5,
  6, 7, 8, 11, 14 post treatment

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Neutropenia is the Major Dose-Limiting Toxicity
of Docetaxel
Neutropenia Decrease in the number of
neutrophils in the blood
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Docetaxel-Induced Neutropenia Across Strains
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Pharmacokinetic Analysis

• Blood drawn 0, 0.5, 1, 2 and 5 hours
  post-injection of 20 mg/kg docetaxel
• 3 animals analyzed per time point
• Docetaxel levels in peripheral blood analyzed via
  LC/MS/MS

Area Under the Curve (AUC)
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Docetaxel AUC Across Strains
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Differential Pharmacodynamics Amongst Strains
Pharmacodynamic Phenomenon
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• The clinical problem
• Multiple active regimens for the treatment of
  most diseases
• Variation in response to therapy
• Unpredictable toxicity

With choice comes decision