1. Pharmacogenetics and Pharmacogenomics
Kevin Zbuk, MD
Medical Oncologist
Juravinski Cancer Centre
McMaster University

2. Outline Introduction and definitions Basic concepts Case studies
Conclusions

3. Pharmacogenetic versus Pharmacogenomic
No universally accepted definitions of either
Often used interchangeably
Pharmacogenetics used for more than 40 years to denote the science about how heritability affects the response to drugs.
Pharmacogenomics is new science about how the systematic identification of all the human genes, their products, interindividual variation, intraindividual variation in expression and function over time affects drug response/metabolism etc.
The term pharmacogenomics was coined in connection with the human genome project
Most use pharmacogenetics to depict the study of single genes and their effects on interindividual differences in (mainly) drug metabolising enzymes, and pharmacogenomics to depict the study of not just single genes but the functions and interactions of all genes in the genome in the overall variability of drugs response

6. Pharmacogenetics
“Pharmacogenetics is the study of how genetic variations affect the disposition of drugs, including their metabolism and transport and their safety and efficacy”
J. Hoskins et. al NRC 2009

7. Pharmacogenetics involves both PK and PD
Pharmacokinetic
“The process by which a drug is absorbed, distributed, metabolized, and eliminated by the body”
Pharmacodynamic
“the biochemical and physiological effects of drugs and the mechanisms of their actions”

8. Goals of Pharmacogen(etics)omics
Maximize drug efficacy
Minimize drug toxicity
Predict patients who will respond to intervention
Aid in new drug development

9. The Hope of Pharmacogenomics
Individuals genetic makeup with allow selective use of medications such that
Efficacy maximized
Side effect minimized

10. This is the hope/hype

11. In the Beginning Mendelian genetics “single gene – single disease”
single wild type allele and single disease allele
Patterns of inheritance included autosomal dominant (need only one disease allele) and autosomal recessive (need two disease alleles)
Followed soon thereafter by additive (co-dominant) model
Both alleles contribute to phenotype

12. Dominant/Recessive

13. Co-dominance

14. Empiric observations suggesting Pharmacogenetics important
Clinical response to many drugs varies widely amongst individuals
Same drug-> same dose -> same indication in different individuals
Some respond
Some don’t
Some don’t respond and have serious toxicity

15. EARLY PK EXAMPLES

16. The beginning of pharmacogenetics
“Inheritance might explain variation in individuals response and adverse effects from drugs” Motulsty
“Pharmacogenetics defined as “study of role of Genetics in drug response” Vogel
Most of studies for next several decades of “high penetrance monogenic” gene-drug interactions
Def: Monogenetic disease. Mutation at single locus sufficient to result in disorder

17. Penetrance
Penetrance of a disease-causing mutation is the proportion of individuals with the mutation who exhibit clinical symptoms.
Eg. if a mutation in the gene responsible for a particular autosomal dominantdisorder has 95% penetrance, then 95% of those with the mutation will develop the disease, while 5% will not.

18. Victor McKusick
Established Online Mendelian Inheritance in Man in early 80s
Categorized majority of Mendelian Disorders
Became very clear that there are many different disease alleles for many disorders (allelic heterogeneity)
Recently many disorders have associated modifier genes that modify disease phenotype
Eg. Age-of-onset and severity

20. Example 1- Success of Pharmacogenetics in Oncology
TPMT

21. TPMT
Main metabolizer of chemotherapeutic agents 6MP and azothiopurine (used mainly in blood based malignancies)
TPMT deficiency leads to severe toxicity associated with treatment (potential mortality)

22. TPMT enzyme activity distribution

23. Hematologic toxicity according to TPMT genotype

24. Evans Nature Reviews Cancer 2006

25. FDA approved pharmacogenetic tests
Drug
Consequence
TPMT
6MP
Toxicity
CYP2D6
Tamoxifen
Decreased efficacy
UGT1A1
Irinotecan
Codeine
Ineffective analgesia
These genes all modulate Pharmokinetics

26. Contribution of High Penetrance Monogenic Model to PG
Contribution likely not as large as initially anticipated
For most pharmacologic traits might be 15-20% at most
Could consider this penetrance
Redundancy likely a major contributing factor
MANY ENZYMES INVOLVED IN DRUG METABOLISM WITH MANY ALTERNATE PATHWAYS
Dichotomous disease versus quantitative trait
Much more likely polygenic model with gene-environment interactions

27. Some of it ain’t genetic
Age
Co-morbidities
Renal and hepatic function (dysfunction)
Concomitant medications
Diet and smoking

28. Common Disease Common Variant Hypothesis
Most complex diseases are strongly influenced by combination of frequent alleles that each only exert modest effect
Polygenic Model (lnheritance)

30. Approach to polygenic pharmacogenomic traits

31. Polygenic Model and PG
Elucidation unlikely possible before advances in genomics
Technologic advances
High throughput sequencing of DNA
Affordable genotyping of 100ks to 1-2M SNPs
Genomic knowledge advances:
Especially Human Genome Project and HapMap Projects

39. Cost of Genotyping In 2005 (5 years ago!) 2009 2014
$1600 to genotype 250K SNPs in one individual
2009
$250 to genotype >1Million SNPs
2014
-$ to genotype >5 millions SNPs

43. Hapmap project There are an estimated 10 million SNPs with MAF >1%
Hapmap project genotyped Chinese, Japanese, African and European individuals (families)

44. HapMap International Consortium
HapMap Project
Phase 1
Phase 2
Phase 3
Samples & POP panels
269 samples
(4 panels)
270 samples
1,115 samples
(11 panels)
Genotyping centers
HapMap International Consortium
Perlegen
Broad & Sanger
Unique QC+ SNPs
1.1 M
3.8 M
(phase I+II)
1.6 M (Affy 6.0 & Illumina 1M)
Reference
Nature (2005) 437:p1299
Nature (2007) 449:p851
Draft Rel. 3
(2010)

46. A more in depth look at PK in clinical practice
Tamoxifen use and CYP2D6

47. Tamoxifen metabolism Needs to be converted to endoxifen to be active
catalysed by the polymorphic enzyme cytochrome P450 2D6 (CYP2D6)
6-10% European population deficient in this enzyme
Efficacy of tamoxifen likely low in this population
Suggests consider alterative treatments

48. J. Hoskins et. al NRC 2009

49. About the CYPs Membrane bound enzymatic proteins
Involved in oxidation, peroxidation and reductive metabolism
Responsible for >90% of drug transformation
Greater than 50 different CYP genes encoding 50 different proteins
CYP2D6 present mainly in liver and a major player in drug metabolism from antidepressants to antihypertensive to chemotherapy

50. Evolution of CYP nomenclature
Initially astute clinical observation of unusual drug response
Such responses then found to be heritable
Early example of phenotype to genotype approach
CYP2D6 polymorphism the first described
Increasing recognition of poor metabolizer phenotype occurred at time that genotyping technology in evolution

51. About CYP2D6
P arm
Location 22q 13.1
Q arm

52. CYP2D6 alleles There are >70 described in this gene
Bottom line: variants either cause no change, decrease somewhat, or significantly decrease metabolism
Extensive metabolizers ( EM), intermediate (IM) metabolizers, and poor metabolizers (PM)
EM is the standard metabolism allele against which others are compared (consider it the wild type)

53. Hoskins et al. Nature Reviews Cancer 2009

54. CYP2D6 alleles Copy Number Variation
Throughout the genome there are areas of DNA that are represented in variable copies in individuals (CNV)
CYP2D6 is one such area
Up to 16 copies seen in some individuals
“NORMAL VARIANT”
ULTRARAPID METABOLIZERS

55. Consequence of CYP2D6 alleles?
EM/EM or EM/IM(PM) normal metabolizers
IM/IM or IM/PM intermediate metabolizers
PM/PM poor metabolizers
Poor/(Intermediate) metabolizers have much lower levels of endoxifen than intermediate/ rapid metabolizers

56. CYP2D6 Genotype and clinical outcomes
Several (small trials) have suggested decreased efficacy of Tamoxifen in poor (intermediate) metabolizers both in adjuvant therapy and in treatment of metastatic disease (see Hoskins NRC 2009 for details)
All retrospective
Largest was only statistically significant association in univariate analysis
In additions several trials have not confirmed these results

57. Reasons for discordant results in CYP2D6 trials
Did not genotype many of the rarer poor metabolizer alleles
Did not account for concurrent use of other drugs metabolized by CYP2D6 in many cases
Different dose of Tamoxifen in several trials
Did not assay endoxifen levels
Power (poor metabolizers rare)
Unknown variants in other genes whose products involved in tamoxifen metabolism

58. So what is needed to clarify the issue of relevance of CYP2D6 genotype and clinical relevance?
Large randomized trial that compares standard dosing of tamoxifen to genotype adjusted dosing
Until that point clinical utility of testing (commerically available) unclear
Should recommend avoiding SSRIs that inhibit CYP2D6 significantly (see later)

59. Provocative thoughts
In post-menopausal breast cancer tamoxifen is falling out of favor due to the efficacy of Aromatase Inhibitors (inhibit extragonadal production of estrogen)
AI shows increased efficacy c/w tamoxifen
BUT MUCH MORE EXPENSIVE AND DIFFERENT S/E PROFILE
Some suggestion that increased efficacy of AI completely explained by decreased efficacy of Tamoxifen in CYP2D6 IM and PM
Punglia (2008) JNCI

60. More relevant to pre-menopausal woman
Can’t use AI alone
In poor metabolizer could consider
Increased dose???
Alternative estrogen receptor modulator not metabolized by CYP2D6 (eg. raloxifen)
Consider AI with ovarian ablation (chemical or otherwise)

61. Ethnic Differences in IM and PM of CYP2D6
PM alleles more common in European population
IM alleles much more common in East Asian and African population
In East Asians Intermediate Metabolizers show similar in vitro CYP2D6 activity c/w Poor Metabolizers in European populations
Gene-gene or gene-environment interactions

62. Drug Co-administration
Antidepressant use common in breast cancer patients
Depression more common in breast cancer patients and antidepressant often used to treat how flashes associated with tamoxifen use
SSRIs (eg. Fluoxetine and paroxetine) inhibit CYP2D6
Level of inhibition varies between different drugs with paroxetine having most inhibition and venlafaxine causing none
Kelly et al. BMJ 2010
Population based cohort study of women receiving tamoxifen adjuvantly for treatment breast cancer
Mortality from breast cancer increased in group using paroxtetine concurrent with tamoxifen

63. Irinotecan – PK example in Colon Cancer
Excreted after conjugation (glucuronidation) by UGT1A1
TATA element (consists of TA repeats) in UGT1A1 promoter shows correlation with transcription levels
More repeats lower transcription levels
An example of a non-SNP variant with clinical relevance
Homozygosity for 7-repeat allele, also known as UGT1A1*28 associated with severe toxicity (diarrhea and low WBC counts mainly)
Results have been somewhat inconsistent but meta-analysis confirms same especially with higher doses of Irinotecan
Homozygosity only in 5-15% of individuals

64. PD example in Colon Cancer Treatment
EGFR inhibitors used in treatment of advanced colon cancer (eg. Cetuximab)
Tumors with k-RAS (and probably BRAF) mutations will NOT respond to EGFR inhibition
Nature Rev. Cancer July 2009

65. Review Paper by Pare et al.

66. Effect of Clopidogrel as Compared with Placebo on Clinical Outcomes among Patients with Acute Coronary Syndromes in the CURE trial, Stratified According to Metabolizer Phenotype.
Figure 1 Effect of Clopidogrel as Compared with Placebo on Clinical Outcomes among Patients with Acute Coronary Syndromes in the CURE trial, Stratified According to Metabolizer Phenotype. Hazard ratios for clopidogrel as compared with placebo are shown for efficacy and bleeding outcomes according to metabolizer phenotype. The size of each symbol is in inverse proportion to the standard deviation of the effect-size estimates. Analyses were performed on data from patients of European or Latin American ancestry, with adjustment for age, sex, and ancestry. Patients with two *2 or *3 alleles (i.e., *2/*2, *2/*3, or *3/*3) were classified as having the poor-metabolizer phenotype, those with one *2 or *3 allele (i.e., *1/*2 or *1/*3) were classified as having the intermediate-metabolizer phenotype, those without a *2, *3, or *17 allele (i.e., *1/*1) were classified as having the extensive-metabolizer phenotype, those with a single *17 allele (i.e., *1/*17) and *17 homozygotes were classified as having the ultrametabolizer phenotype, and those with one *17 allele and one loss-of-function allele (i.e., *2/*17 or *3/*17) were classified as having an unknown metabolizer phenotype. Only patients who were successfully genotyped for all three single-nucleotide polymorphisms were included in these analyses.
Paré G et al. N Engl J Med 2010;363:

67. Kaplan–Meier Curves for Event-free Survival According to CYP2C19 Loss-of-Function and Gain-of-Function Allele Carrier Status among European and Latin American Patients with Acute Coronary Syndromes in the CURE Trial.
Figure 2 Kaplan–Meier Curves for Event-free Survival According to CYP2C19 Loss-of-Function and Gain-of-Function Allele Carrier Status among European and Latin American Patients with Acute Coronary Syndromes in the CURE Trial. Loss-of-function allele carriers were defined as patients with at least one loss-of-function allele (i.e., *2 or *3): *1/*2, *1/*3, *2/*2, *2/*3, *3/*3, *2/*17, or *3/*17; loss-of-function noncarriers were defined as patients with no loss-of-function allele: *1/*1, *1/*17, or *17/*17. Gain-of-function carriers were defined as carriers of at least one gain-of-function allele (i.e., *17): *1/*17, *17/*17, *2/*17, or *3/*17; gain-of-function noncarriers were defined as patients with no gain-of-function allele: *1/*1, *1/*2, *1/*3, *2/*2, *2/*3, or *3/*3.
Paré G et al. N Engl J Med 2010;363:

68. Effect of Clopidogrel as Compared with Placebo on Clinical Outcomes among Patients with Atrial Fibrillation in ACTIVE A, Stratified According to Metabolizer Phenotype.
Figure 3 Effect of Clopidogrel as Compared with Placebo on Clinical Outcomes among Patients with Atrial Fibrillation in ACTIVE A, Stratified According to Metabolizer Phenotype. Hazard ratios for clopidogrel as compared with placebo are shown for efficacy and bleeding outcomes according to metabolizer phenotype. The size of each symbol is in inverse proportion to the standard deviation of the effect-size estimates. Analyses were performed on data from patients of European ancestry, with adjustment for age and sex. Patients with two *2 or *3 alleles (i.e., *2/*2, *2/*3, or *3/*3) were classified as having the poor-metabolizer phenotype, those with one *2 or *3 allele (i.e., *1/*2 or *1/*3) were classified as having the intermediate-metabolizer phenotype, those without a *2, *3, or *17 allele (i.e., *1/*1) were classified as having the extensive-metabolizer phenotype, those with a single *17 allele (i.e., *1/*17) and *17 homozygotes were classified as having the ultrametabolizer phenotype, and those with one *17 allele and one loss-of-function allele (i.e., *2/*17 or *3/*17) were classified as having an unknown metabolizer phenotype. Only patients who were successfully genotyped for all three single-nucleotide polymorphisms were included in these analyses.
Paré G et al. N Engl J Med 2010;363:

69. Kaplan–Meier Curves for Event-free Survival According to CYP2C19 Loss-of-Function and Gain-of-Function Allele Carrier Status among European Patients with Atrial Fibrillation in ACTIVE A.
Figure 4 Kaplan–Meier Curves for Event-free Survival According to CYP2C19 Loss-of-Function and Gain-of-Function Allele Carrier Status among European Patients with Atrial Fibrillation in ACTIVE A. Loss-of-function allele carriers were defined as patients with at least one loss-of-function allele (i.e., *2 or *3): *1/*2, *1/*3, *2/*2, *2/*3, *3/*3, *2/*17, or *3/*17; loss-of-function noncarriers were defined as patients with no loss-of-function allele: *1/*1, *1/*17, or *17/*17. Gain-of-function carriers were defined as carriers of at least one gain-of-function allele (i.e., *17): *1/*17, *17/*17, *2/*17, or *3/*17; gain-of-function noncarriers were defined as patients with no gain-of-function allele: *1/*1, *1/*2, *1/*3, *2/*2, *2/*3, or *3/*3.
Paré G et al. N Engl J Med 2010;363:

70. Baseline Characteristics of Genotyped Patients in the CURE and ACTIVE A Trials.
Table 1 Baseline Characteristics of Genotyped Patients in the CURE and ACTIVE A Trials.
Paré G et al. N Engl J Med 2010;363:

71. Why is pharmacogenomics not widely utilized in the clinic
It required a shift in clinician attitude and beliefs “not one dose fits all”
Paucity of studies demonstrating improved clinical benefit from use of pharmacogenomic data
Still much to be learned
Even some of the black block warnings currently on drug labels may be overcalls of importance
Genome wide interrogation will likely be important to get the entire picture

72. Conclusion
Genetic variation contributes to inter-individual differences in drug response phenotype at every pharmacologic step
Through individualized treatments, pharmacogenetics and pharmacogenomics are expected to lead to:
Better, safer drugs the first time
More accurate methods of determining appropriate drug dosages
Pharmacogenomics offers unprecedented opportunities to understand the genetic architecture of drug response
HOWEVER IN MANY CASES NOT YET READY FOR PRIME TIME!!!