Pharmacogenetics

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Pharmacogenetics Chemotherapy

• Jane S. Chawla, M.D.
• January 30, 2009

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Over the next 30 mintues

• Intro to Pharmacogenetics
• Some common polymorphisms
• DPD
• UGT1A1
• TPMT
• CYP2D6
• Conclusions

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Pharmacogenetics Plays a Role in Drug Toxicity
Efficacy
Many sources of interindividual variation in drug
toxicity and efficacy, both pharmacokinetic and
pharmacodynamic.
Deeken, J et al. Anti-cancer drugs, 2007.
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Genetic Polymorphisms

• Every gene contains some degree of polymorphism
• Single nucleotide polymorphism (SNPs) occur every
  1000-3000 base pairs throughout the genome
• Sometimes difficult to determine which are
  relevant to treatment of disease
• Polymorphisms can occur in
• Drug targets (including cell surface receptors
  and target proteins)
• Hormonal-regulated enzymes
• Genes involved in drug pharmacokinetics that
  impact drug absorption, distribution, metabolism,
  and excretion
• Genetic polymorphisms can lead to
• Severe toxicity when drugs not metabolized
  normally
• Diminished or increased therapeutic affect

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5-Fluorouracil DPD Deficiency
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5-Fluorouracil Catabolism

• DPD (Dihydropyrimidine dehydrogenase) is the 1st
  and rate-limiting enzyme in the pathway involved
  in the degradation of pyrimidine bases uracil and
  thymine
• DPD is responsible for catabolism of
  5-fluorouracil
• This accounts for gt85 of drug metabolism

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5-FU-induced Toxicity

• Inability to inactivate 5-FU can lead to
  neurological, hematopoietic, and GI toxicities
  that can be fatal
• It is reported that about 31 of pts with
  advanced colorectal cancer who receive bolus 5-FU
  regimens have grades 3-4 hematologic toxicity
• It is estimated that 40-60 of cancer pts with
  severe 5-FU toxicity have DPD-deficiency

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DPD Deficiency

• Variance in DPD is a common genetic polymorphism
• 39 different mutations / polymorphisms identified
• 14 of these in pts with excessive 5-FU toxicity
• 3-5 of Caucasians heterozygous for DPD mutations
• 0.1 of Caucasians homozygous for DPD mutations
• DPD activity varies 8-21-fold among individuals

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IVS14 1GgtA is the Most Common DPD Polymorphism

• G?A transition at the 5 splice consensus
  sequence of exon 14
• Exon 14 is deleted ? translation of
  non-functional enzyme
• This polymorphism accounts for 50 of known
  nonfunctional DPD alleles

Deeken, J et al. Anit-Cancer Drugs, 2007.
Nature Reviews Cancer, 2001
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Ethnic Frequency of DPD Deficiency IVS141GgtA
Variant

• DPD deficiency seen in
• Chinese, Indian, Malay, Japanese, Korean pts
• DPD enzyme activity is lower in healthy African
  Americans compared to Caucasians
• 8 versus 2.8

Ethnic frequency of IVS141GgtA variant
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Is ? DPD Activity Predictive of Severe
5-FU-related Toxicity?

• 131 Caucasian pts with severe 5-FU toxicity
• DPD activity lower in pts with 5-FU toxicity
  compared to general cancer population (13 vs
  2.7)
• The lower the DPD activity, the greater the
  grades of toxicity
• DPD deficiency only partially explains
  5-FU-related toxicity
• Of the 9 with lethal toxicity, 2 pts had normal
  DPD activity
• Weak negative correlation b/w global toxicity
  score DPD activity
• IVS141GgtA variant found in only 2 pts (2.2),
  both with ?? toxicity

DPD Activity and Toxicity in 131 Patients Treated
with 5-FU
Magné et al. Br J of Clin Pharm, 2007.
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Should We Test?

• Screening for genetic mutations alone is not
  likely to effectively identify pts susceptible to
  lethal 5-FU toxicity
• Genotype alone does not predict which pts will
  develop toxicity
• Of Caucasians with DPD deficiency, only about 50
  have the most common IVS141GgtA variant
• Peripheral blood testing for DPD activity in
  mononuclear cells is difficult and not conducive
  to wide use
• Evidence of a circadian rhythm affecting DPD
  levels
• About 50 of patients with severe 5-FU-related
  toxicity are NOT DPD deficient!
• DPD deficiency only partially explains
  5-FU-related toxicity
• Hypermethylation of DPYD promoter is associated
  with down-regulation of DPD activity in patients
• Work being done on a rapid noninvasive 2-¹³C
  uracil breath test to determine DPD activity

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FDA Label Recommendations

• FDA mentions DPD deficiency as a cause of severe
  toxicity
• No specific recommendations about dose-reductions
  or discontinuing the drug
• No recommendations about genetic testing

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Irinotecan UGT1A1
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Irinotecan Metabolism
Competition for substrate b/w Activation
inactivation pathways
SN-38 activation Required for anti- Tumor effect
? To bile
1000x more Active than Irinotecan
UGT1A1 inactivates SN-38 by glucuronidation via
UDP glucuronosyltransferases
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Polymorphisms in the UGT1A1 gene/promoter

• 60 different genetic variations in UGT1A1 gene
  and promoter
• Familial forms of hyperbilirubinemia due to
  UGT1A1 polymorphism
• Crigler-Najjar type 1
• Gilberts syndrome (3-10 of population)
• Some genotypes associated with irinotecan
  toxicity
• Promoter region of UGT1A128
• Coding regions of UGT1A16, UGT1A127, UGT1A129,
  UGT1A17
• Ethnic variation
• Caucasians African Americans ? variation in the
  promoter region
• Asians ? missense mutations in the coding region

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UGT1A1
UGT1A1 promoter Contains TA repeats
Wild type has 6 TA repeats
7 TA repeats ? UGT1A1 enzyme expression
Glucuronidation makes SN-38 more soluble
allows it to be eliminated in bile
? inactivation of SN-38
Nature Reviews Cancer, 2001
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Is the Presence of a UGT1A1 Polymorphism
Predictive of Toxicity?

• Case-control, retrospective study by Ando et al
• Presence of UGT1A1 polymorphisms evaluated in
  Japanese pts who previously received irinotecan
• Severe toxicity grade 4 leukopenia and/or grade
  3 or 4 diarrhea
• Of the 26 with severe toxicity
• 15 homozygous for UGT1A128
• 31 heterozygous for UGT1A128
• Of the 92 without severe toxicity
• 3 homozygous for UGT1A128
• 11 heterozygous for UGT1A128
• Those with UGT1A128 were 7x more likely to
  encounter toxicity
• The frequency of the UGT1A128 allele was
  3.5-fold higher in those pts with grade 4
  leukopenia and/or grade 3 or 4 diarrhea

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UGT1A128 Polymorphism Associated with Decreased
Glucuronidation of SN-38

• Prospective study of 20 Caucasian pts treated
  with irinotecan for solid tumor malignancies
• UGT1A128 polymorphism
• 45 ? WT (TA6/TA6)
• 35 ? Heterozygous (TA6/TA7)
• 20 ? Homozygous (TA7/TA7)
• Hetero / homozygous pts had ? glucuronidation of
  SN-38 ? SN-38 levels (3.9-fold lower)
• Hetero / homozygous pts had ? grades of diarrhea
  neutro-penia (2.5-fold lower ANC post-tx)

Iyer, L et al. The Pharmacogenetics Journal, 2002.
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Does UGT1A1 Genotyping Improve Outcomes?

• Evidence-based review of outcomes in CRC pts
  treated with irinotecan found
• Genetic testing has high sensitivity /specificity
  for the common genotypes
• No prospective studies examining rate of adverse
  events in pts given dose reduced irinotecan
• No direct evidence to show that dose
  modifications based on UGT1A1 genotype benefits
  or harms CRC pts
• Homozygous UGT1A128 pts had improved survival
  statistically significant higher tumor response
  rates

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FDA Label Recommendations

• Individuals who are homozygous for the UGT1A128
  allele are at increased risk for neutropenia
• FDA recommends a reduction in the starting dose
  in pts known to be homozygous for UGT1A128
• Precise dose reduction is not known and should
  be considered based on individual patient
  tolerance to treatment
• No recommendations for dose-reductions for
  hetero-zygous patients

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Mercaptopurine TPMT
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Thiopurines Thiopurine Methyltransferase (TPMT)

• Thiopurines are purine analogs
• Includes mercaptopurine (6-MP), thioguanine,
  and azathioprine
• 6-MP has been used in the treatment of childhood
  ALL
• This class of drugs is now used more for
  autoimmune disorders to prevent organ rejection
• TPMT inactivates thiopurines

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6-MP Metabolism
MP is converted to (TGNs) thioguanine
nucleotides then incorporated into DNA
TPMT methylates MP prevents TGN formation
excessive drug toxicity
Antileukemic effects myelosupression
Nature Reviews Cancer, 2001
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TPMT Polymorphisms Lead to Reduced Enzyme Activity

• Variability between individuals in erythrocyte
  TPMT activity is a heritable autosomal
  co-dominate trait
• SNPs ? amino acid substitutions ? TPMT enzyme is
  ubiquitylated destroyed
• 8 TPMT alleles have been identified
• TPMT2
• TPMT3A
• TPMT3C
• As the of mutant alleles ?, TPMT activity in
  erythrocytes and leukemic blasts ? TGN
  concentration ?
• Homozygous --gt ?? TPMT activity ?? TGN
• Heterozygous ? ? TPMT activity ? TGN
• Wild-type ? Normal TPMT activity TGN

These 3 account for gt95 of the
clinically- significant mutations resulting in
low or intermediate enzyme activity
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Influence of Ethnicity on TPMT Mutation Frequency
Deeken, J et al. Anti-cancer drugs, 2007.

• Frequency of reduced TPMT activity in
  Caucasians
• 86.6 have high TPMT activity
• 11.1 have intermediate activity
• 0.3 have deficient TPMT activity

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TPMT Polymorphisms Affect 6-MP Therapeutic
Efficacy Toxicity

• TPMT Polymorphism therapeutic efficacy
• Children with median TGN concentration less than
  the population median had worse RFS compared to
  those with concentration greater than the median
• Patients with at least 1 mutant TPMT allele tend
  to have improved response to 6-MP compared to pts
  with both WT alleles
• TPMT Polymorphism risk of toxicity
• Reports of toxicity when full-dose 6-MP given to
  homozygous pts
• As TGN ?, risk of leukopenia ? risk of
  relapse ?
• Dose reductions in homozygous ALL pts ? ?TGN
  ? risk of relapse
• TPMT genotype may influence risk of secondary
  malignancies (brain tumors AML)

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TPMT Genotype Correlates Well With Phenotype
(enzyme activity)
TPMT phenotypes in thiopurine-intolerant
patients compared with the general population

• Study of 23 children with ALL intolerant to 6-MP
• 100 concordance b/w genotype phenotype in the
  homozygous deficient WT patients
• Genotyping for common TPMT alleles can identify
  pts at risk for severe 6-MP toxicity
• Dose reductions enabled re-initiation of therapy
• Median dose? homozygous ? 90.8 heterozygous ?
  67

6-fold overrepresentation of TPMT- Deficient
patients heterozygotes in the
thiopurine-intolerant Group ( Plt0.001)
Evans, WE. J Clin Oncol, 2001.
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How Do We Best Test for Variability in TPMT
Activity?

• Measurement of TPMT activity RBC TGN levels is
  difficult
• Labor-intensive assays
• Laboratory-related variability
• Multiple studies have shown correlation between
  TPMT genotype phenotype
• Type of polymorphism number of affected alleles
  could help to guide dose reductions

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FDA Label Recommendations

• TPMT genotyping or phenotyping suggested for
  patients with severe toxicity
• Dose reduction recommendations
• Substantial dose reductions may be needed for
  homozygous patients to avoid life threatening
  bone marrow suppression
• Specific dose adjustment not recommended
• No dose adjustments recommended for heterozygous
  patients

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Tamoxifen CYP2D6
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CYP2D6 Polymorphisms and Tamoxifen

• Tamoxifen is a prodrug that is converted to its
  active metabolites endoxifen and 4-OH-TAM
• 4-OH-TAM is 30x more active
• Endoxifen is 100x more active
• CYP2D6 metabolizes Tamoxifen to endoxifen
• Over 80 genetic variants of CYP2D6 have been
  described
• Polymorphisms of CYP2D6 may produce a less active
  enzyme
• CYP2D6 polymorphisms may affect response to
  tamoxifen

Dezentjé, V. Clinical Cancer Research, 2009.
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Does CYP2D6 Polymorphism Affect Tamoxifen
Metabolism?

• CYP2D6 polymorphisms affect drug metabolism
• Based on mutation, enzyme activity can be normal
  to absent
• Labeled as poor, intermediate, extensive, or
  ultrarapid metabolizer
• Number of inactive alleles affects metobolism
• Homozygous carriers (Ex 4/4) are poor
  metabolizers
• Heterozygous carriers (Ex 1/4) are either
  intermediate or extensive metabolizers
• There is concern that decreased CYP2D6 activity
  will lead to undertreatment with Tamoxifen

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CYP2D6 Genetic Variants
Dezentjé, V. Clinical Cancer Research, 2009.
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CYP2D6 Genotype Risk of Breast Cancer Recurrence
Dezentjé, V. Clinical Cancer Research, 2009.
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How Do We Interpret These Conflicting Data?

• 6 studies were evaluated in mainly Caucasian
  breast cancer population
• 3 studies ? link b/w CYP2D6 genotype ? risk of
  recurrence
• 2 studies ? no link b/w CYP2D6 genotype
  tamoxifen efficacy
• 1 study ? link b/w CYP2D6 genotype ? risk for
  recurrence
• Confounders
• Heterogeneous populations of pts
• Tumor grade, Her-2 status, pre/postmenopausal,
  tamoxifen dose tx length
• Where to analyze data from heterozygous pts (w/
  WT or homozygous pts)?
• No control for CYP2D6 inhibitors (SSRIs used in
  up to 30 of pts)
• ? compliance in extensive metabolizers from side
  effects

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Conclusions

• Screening for these polymorphisms prior to
  initiating treatment is not recommended
• Screening for DPD, UGT1A1, and TPMT polymorphisms
  can be done in the setting of severe toxicity
• Most cancer pts are treated with a
  trial-and-error approach to adjustments in
  dosage
• Most patients with severe toxicity will require
  dose adjust-ments regardless of presence /
  absence of polymorphism

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Conclusions

• Need more prospective studies examining affect of
  dose adjustments on patients with polymorphisms
  that alter drug metabolism
• Does dose-reduction improve outcomes in patients
  with polymorphisms?
• Delicate balance between decreasing toxicity with
  dose-reduction and increasing risk of relapse
• As our understanding of the relationships between
  genotype phenotype increases, we may be able to
  treat our patients with a more tailored approach

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Resources

• Wei, X. et al. Molecular Basis of the Human
  Dihydropyrimidine Dehydrogenase Deficiency and
  5-Fluorouracil Toxicity. The Journal of Clinical
  Investigation 1996, 98 610-615.
• Ando, Y et al. Polymorphisms of
  UDP-Glucuronosyltransferase Gene and Irinotecan
  Toxicity A Pharmacogenetic Analysis. Cancer
  Research 2000, 60 6621-6926.
• Iyer, L. et al. UGT1A128 polymorphism as a
  determinant of irinotecan disposition and
  toxicity. The Pharmacogenomics Journal 2002, 2
  43-47.
• Innocenti, F. et al. Genetic Variants in the
  UDP-glucuronosyltransferase 1A1 Gene Predict the
  Risk of Severe Neutropenia of Irinotecan.
  Journal of Clinical Oncology 2004, 22 1382-88.
• Dezentjé, V. et al. Clinical Implications of
  CYP2D6 enotyping in Tamoxifen Treatment for
  Breast Cancer. Clinical Cancer Research 2009,
  15 15-21.
• Magné, N. et al. Dihydropyrimidine dehydrogenase
  activity and the IVS141GgtA mutation in patients
  developing 5FU-related toxicity. British Journal
  of Clinical Pharmacology 2007, 64 237-240.
• Mattison, L.K. et al. The uracil breath test in
  the assessment of dihydropyrimidine dehydrogenase
  activity pharmacokinetic relationship between
  expired 13CO2 and plasma 2-13Cdihydrouracil.
  Clinical Cancer Research 2006, 15 549-55.
• Mattison, LK. Increased prevalence of
  dihydropyrimidine dehydrogenase deficiency in
  African-Americans compared with Caucasians.
  Clinical Cancer Research 2006, 12 5491-5.
• Evans, W.E. Preponderance of Thiopurine
  S-Methyltransferase Deficiency and Heterozygosity
  Among Patients Intolerant to Mercaptopurine or
  Azathioprine. J of Clinical Oncology 2001, 19
  2293-2301.
• Relling, MV et al. Pharmacogenetics and Cancer
  Therapy. Nature Reviews Cancer 2001, 1
  99-108.
• Maitland, ML et al. TPMT, UGT1A1, and DPYD
  genotyping to ensure safer cancer therapy?
  Trends in Pharmacological Sciences 2006, 27
  432-437.
• Deeken, JF et al. Toward Individualized
  Treatment Prediction of Anticancer Drug
  Disposition and Toxicity with Pharmacogenetics.
  Anti-Cancer Drugs 2007, 18 111-126.

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