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Introduction
Our current approach to drug therapy is largely empiric
and based on clinical studies that define the maximally
tolerated dose and reasonable toxicity in a narrowly defined
population. This approach typically leads to the safe and
effective administration of drugs to most individuals.
However, empiric therapy is more likely to lead to poor
responses or exaggerated toxicities in patients receiving
drugs with a small therapeutic window, such as anti-cancer
drugs. Tailoring therapy using genetic information is one
means to improve response and tolerability to anti-cancer
drugs.
Clinical pharmacogenomics is the study of the human
genome for understanding the variability in response to
drug therapy. Genetic variations may explain in part some
of the well-documented variability of response to anticancer drugs. Polymorphisms are genetic variations that
occur in at least 1% of the population. The most common
polymorphisms are single nucleotide polymorphisms, which
occur when one nucleotide base replaces another nucleotide
base within the DNA sequence. These polymorphisms may
be synonymous or nonsynonymous; a nonsynonymous
Pharmacotherapy Self-Assessment Program, 6th Edition
Tamoxifen
Tamoxifen is a selective estrogen receptor modulator
commonly administered to women with hormone receptor
positive breast cancer after surgery with or without adjuvant
chemotherapy. The optimal duration of therapy remains
poorly defined, but 5 years of therapy can reduce the relative
risk of recurrence by almost 50%, and the benefits last for
39
Pharmacogenomics
Abbreviations in
This Chapter
ALL
AML
ATP
CML
CYP
DPD
EGFR
FISH
IHC
KRAS
SSRI
SULT
TPMT
UGT
40
Clinical Testing
In 2006, the FDA supported the inclusion of data
regarding an association between the CYP2D6 genotype
and an increased risk of disease recurrence in the labeling
of tamoxifen; however, no consensus was reached regarding
a recommendation for genetic testing. A DNA microarray
currently available from Roche is the first FDA-cleared in
vitro diagnostic test for CYP2C19 and CYP2D6. Genotype
information for 29 alleles is compiled to predict an
individuals metabolic capacity based on published reports.
The specificity of the test is 100% for the wild-type alleles,
and the sensitivity is 99% for CYP2D6 and CYP2C19 alleles.
The genotype is reported as an extensive, intermediate,
or poor metabolizer. This microarray could be used to
determine CYP2D6 metabolic capacity before administering
tamoxifen, if prospective studies ultimately indicate that
determining a womans genotype before receiving tamoxifen
improves long-term outcomes.
Although data support a relationship between genotype
and recurrence, no prospective study provides evidence that
genotyping before initiating tamoxifen therapy improves
outcomes compared with no genotyping. Furthermore, it is
not known whether genetic testing will improve outcomes in
postmenopausal women receiving tamoxifen compared with
women receiving aromatase inhibitors. Additional concerns
stem from the limited genotypes examined retrospectively;
more than 70 variant CYP2D6 alleles have been identified
to date, and although CYP2D6 substrate specificity is
dependent on race, the inclusion of different ethnicities in
these analyses has been limited.
41
Pharmacogenomics
Irinotecan
Clinical Testing
In 2005, irinotecan product information was revised
to include a description of the prevalence of UGT1A1
polymorphisms, the increased risk of neutropenia with these
polymorphisms, and a recommendation to start treatment
with a lower dose in these patients. A one-level dose
reduction (about a 20% dose reduction) is recommended for
patients homozygous for the UGT1A1*28 allele. In August
2005, the FDA approved a pharmacogenetic test for UGT1A1.
This test detects and identifies mutations in the UGT1A1
gene. The test can be used as an aid to individualize the
dose of irinotecan for a specific patient; however, it is not
clear how this information should be used. Considerations
regarding therapy should include treatment intent (curative
vs. palliative), alternative therapies, and clinical evidence
regarding the outcomes associated with lower doses.
Furthermore, the racial distribution in studies is not always
provided within the publications; thus, it is not known if this
association is applicable to all races.
Fluorouracil
Fluorouracil is an integral part of chemotherapy regimens
for gastrointestinal, breast, cervical, and head and neck
cancers. The most common grade 3 or 4 toxicities include
hematologic, dermatologic, and gastrointestinal toxicities.
Fluorouracil is a pyrimidine analog that is inactivated by
the polymorphic enzyme dihydropyrimidine dehydrogenase
(DPD); this catabolism is the initial and rate-limiting step
in the inactivation of fluorouracil and metabolizes more
than 80% of fluorouracil. Its oral analog, capecitabine,
also is inactivated by DPD. The remaining portion of the
drug undergoes metabolism to form a monophosphate,
which forms a complex that impairs DNA synthesis and
is responsible for the antiapoptotic and cytotoxic effects
associated with fluorouracil.
42
DPD Polymorphisms
The catalytic activity of DPD varies by at least 20fold, and low activity substantially limits the inactivation
of fluorouracil. More than 55 genetic variations, including
synonymous and nonsynonymous single nucleotide
polymorphisms and gene deletions, have been identified;
however, most variations do not have functional
consequences. The most common polymorphism, DYPD*2A,
accounts for 40% to 50% of patients with reduced or no DPD
activity. The polymorphism leads to the transcription of a
nonfunctional protein. Subsequently, the elimination halflife of fluorouracil is increased in patients heterozygous for
the DYPD*2A allele. Initial data suggest that DPD deficiency
may be associated with improved response or decreased
tolerability; however, a molecular basis for reduced activity
is only defined in about 57% of patients.
Sex and racial differences in DPD activity have been
reported. Women experience greater toxicities compared
with men after receiving fluorouracil; it appears that DPD
activity in the tumor tissue is lower in women compared
with men. Reduced DPD activity is reported in 3% to 5% of
whites and in 8% of African Americans. Furthermore, mean
DPD activity is lower in African Americans and higher in
Asians compared with whites. Evaluation of the literature
also indicates that single nucleotide polymorphisms and
haplotype distributions are dependent on race and ethnicity.
However, these comparisons should be interpreted with
caution because no consensus exists regarding the definition
of deficiency.
Mercaptopurine
Mercaptopurine is an oral purine analog commonly
prescribed to patients with acute lymphoblastic leukemia
(ALL) in combination with methotrexate as part of
maintenance therapy; its dose-limiting toxicities are
neutropenia and anemia.
Mercaptopurine undergoes metabolism by a series of
nucleotide enzymes to several phosphorylated metabolites
that are eventually incorporated into RNA and DNA.
The cytosolic enzyme thiopurine methyltransferase
(TPMT) catalyzes the inactivation of mercaptopurine and
43
Pharmacogenomics
TMPT Testing
Both phenotyping and genotyping methods may be used
to identify TPMT deficiency. Phenotyping assays include
ex vivo measurement of thioguanine nucleotides or TPMT
activity in erythrocytes. Thiopurine methyltransferase
activity may be measured using radiolabeled substrates,
high-performance
liquid
chromatography,
and
radioimmunoassays. Limitations include poor concordance
among the different methods for measuring thioguanine
nucleotides or TPMT activity in erythrocytes; one study
suggested that up to a 2.6-fold difference in TPMT activity
measurement is caused by the methodology. Red blood cell
transfusions and concurrent drug therapy with sulfasalazine
and other aminosalicylate drugs may also affect these
methods. Moreover, the underlying disease may influence
enzymatic activity; one study indicated that TPMT activity
was higher in blasts from patients with AML compared with
blasts from patients with ALL, making the definition of
TPMT deficiency dependent on the disease state. However,
TPMT activity still corresponded with genotype in these
populations.
44
Epidermal Growth
Factor Inhibitors
Three anti-cancer drugs are classified as epidermal
growth factor receptor (EGFR) inhibitors: erlotinib,
cetuximab, and panitumumab. Erlotinib is a small molecule
that inhibits the EGFR tyrosine kinase by binding to the
adenosine triphosphate (ATP)-binding site of the receptor.
On stimulation, two EGFRs form a homodimer and
autophosphorylate each other with a molecule of ATP.
Autophosphorylation leads to a conformational change that
exposes protein-binding sites and subsequently initiates
an intracellular signaling cascade. Erlotinib suppresses
the intracellular signaling cascades by preventing
autophosphorylation of the receptors.
Erlotinib is used to treat nonsmall cell lung cancers and
head and neck cancers, as well as other solid tumors. Tumor
regression and disease stabilization occurs when it is used
as second-line therapy for patients with advanced nonsmall
cell lung cancer.
Panitumumab and cetuximab are monoclonal antibodies
that also suppress the downstream intracellular signaling
cascades associated with the EGFR. These antibodies block
the binding of endogenous ligands (e.g., epidermal growth
factor, transforming growth factor-alpha) to the EGFR.
These antibodies may also down-regulate receptor levels.
Pharmacotherapy Self-Assessment Program, 6th Edition
45
Pharmacogenomics
Panitumumab
Panitumumab was also exclusively studied in patients
with EGFR-positive tumors using the same commercial
assay incorporated into the lead trials for cetuximab and
recently given a diagnosis of recurrent colorectal cancer
after treatment with fluoropyrimidines, oxaliplatin, and
irinotecan. A recent study indicates that KRAS mutations
predict lack of response to this antibody. The response
rate was 0% for patients with KRAS mutation and 17%
for patients with KRAS wild type. The patients with
KRAS wild type also experience a longer median time to
progression (12.3 months vs. 7.4 months). It appears the
EGFR expression and KRAS wild type are also predictors
of response to panitumumab.
Clinical Testing
Epidermal growth factor receptor testing includes
immunohistochemistry (IHC), FISH, and mutational analyses
of the gene. Although several retrospective studies of these
tests demonstrate a strong predictive value of EGFR gene
expression, prospective validation is warranted to adequately
determine which method will be clinically useful to predict
treatment outcomes in response to erlotinib. Two studies
indicate some discordance among the different methods.
Epidermal growth factor receptor status is measured in
tissue blocks by three methods: (1) protein expression by
standardized IHC, (2) gene copy number by FISH, and (3)
mutation analysis by sequencing. Substantially more tumors
were positive for EGFR using IHC or FISH compared with
mutation analysis. All patients positive for an activating
somatic EGFR mutation experienced a complete or partial
response after receiving gefitinib or erlotinib, although more
than half of these tumors were IHC negative or FISH negative.
In a subsequent study, a poor association between protein
expression and mutation analysis was demonstrated, but a
strong association between gene amplification and mutation
analysis was identified. Both parameters were associated
with improved response and longer time to progression and
overall survival. A retrospective study also demonstrated
a poor correlation between mutation analysis and IHC. A
mass spectrometry assay has also been validated as part of
a multi-institutional study to identify the likelihood of good
or poor outcomes after treatment with erlotinib in patients
with nonsmall cell lung cancer.
Because the indiscriminate use of gefitinib or erlotinib in
patients with nonsmall cell lung cancer produces limited
Cetuximab
Cetuximab was exclusively studied in patients with
metastatic colorectal cancer whose previous therapy with
irinotecan and oxaliplatin failed and whose tumors stained
positive for EGFR using a commercial assay. The percentage
of cells staining positive for EGFR and the signal intensity
were scored. However, the subpopulations of patients
who respond to cetuximab remain poorly defined. Only
one study indicates that patients with a higher gene copy
number by FISH are more likely to respond to cetuximab,
as demonstrated in studies that showed a correlation
between gene copy number and response to erlotinib in
patients with lung cancer. Patients with FISH-positive
tumors experienced a significantly higher response rate and
longer time to progression compared with FISH-negative
tumors. Additional data support that tumors with wildPharmacogenomics
46
BCR-ABL Mutations
The underlying mechanism of resistance to imatinib
includes BCR-ABL gene amplification, BCR-ABL messenger
RNA, and protein overexpression and BCR-ABL point
mutations; these point mutations appear to be the most
common mechanism of resistance to imatinib. The most
common mutations are found in the tyrosine kinase domain,
where they impair the binding of imatinib to this tyrosine
kinase domain. More than 50 point mutations have been
identified; the most common mutations that account for
60% to 70% of the resistance to imatinib include Gly250,
Tyr253, Glu255, Thr315, Met351, and Phe359.
Bone marrow or peripheral blood samples from 370
patients with CML or Philadelphia chromosomepositive
ALL treated with imatinib were assessed for mutations
within this kinase domain. Around 43% of the patients
had at least one mutation. Mutations were more common
in patients in accelerated phase or blast crisis and in
patients with acquired resistance. In addition, mutations
were more common in patients who had received prior
interferon alfa therapy compared with patients who
received imatinib as first-line therapy. The T315I mutation
was more commonly identified in patients with advanced
disease. Furthermore, African Americans with a diagnosis
of Philadelphia chromosomepositive CML tended to have
more chromosomal abnormalities compared with whites,
and the median survival time was shorter in this population.
However, it is not known whether African Americans have
more de novo or acquired mutations in the tyrosine kinase
domain. Sex or additional racial differences have not been
explored.
Clinical Outcomes
These mutations are de novo or acquired and lead to
disease progression and decreased overall survival. One
mechanism to overcome this resistance is to escalate the
dose of imatinib. Previous studies have demonstrated that
patients with chronic-phase CML experiencing hematologic
or cytogenetic resistance to imatinib can experience a
hematologic or cytogenetic response after increasing the
dose of imatinib to 600800 mg/day orally.
Both dasatinib and nilotinib demonstrate sensitivity to
most point mutations, and clinical responses are demonstrated
in clinical trials in some patients with imatinib-resistant
disease. However, differences are noted in the sensitivity
of these drugs to select point mutations. For example, the
Phe317 mutation causes decreased sensitivity to imatinib
and dasatinib but retains sensitivity to nilotinib, and the
Tyr253 mutation causes resistance to imatinib and nilotinib
but retains sensitivity to dasatinib. It appears that increasing
doses of nilotinib or dasatinib may overcome some initial
resistance to these drugs with these select mutations.
Unfortunately, neither dose escalation with imatinib nor
47
Pharmacogenomics
Clinical Testing
Genzyme provides standardized molecular testing
services as part of the CML ALLIANCE. The alliance offers
a quantitative polymerase chain reaction test to measure
BCR-ABL transcript concentrations in the bone marrow or
peripheral blood. The test is promoted to guide therapeutic
decisions regarding response to imatinib. However, this
test does not detect point mutations within the tyrosine
kinase domain. Tests for mutations should be considered
when there is (1) failure to achieve complete hematologic
response at 3 months; (2) no cytogenetic response at 6
months; (3) failure to achieve major cytogenetic response at
12 months; and (4) any sign of loss of response per the most
recent clinical practice guidelines. Several laboratories offer
testing to detect these mutations. Direct sequencing of the
tyrosine kinase domain is completed using bone marrow
aspirate or peripheral blood. Total RNA is extracted, and
the first strand of complementary DNA is synthesized; the
DNA is used for nested polymerase chain reactions that
then undergo direct sequencing. The results usually include
codon number, mutation, and abundance of the identified
mutations.
Conclusion
3.
Goetz MP, Rae JM, Suman VJ, Safgren SL, Ames MM,
Visscher DW, et al. Pharmacogenetics of tamoxifen
biotransformation is associated with clinical outcomes of
efficacy and hot flashes. J Clin Oncol 2005;23:93128.
This was the first study to associate the CYP2D6 genotype
with disease outcome. The study assessed 223 paraffinembedded tumor samples from patients who had received
5 years of adjuvant tamoxifen therapy. Alleles of CYP2D6
(*4 and *6) and CYP3A5 (*3) were evaluated. The primary
outcomes assessed were relapse-free time, disease-free
survival, and overall survival. Women homozygous for the
CYP2D6*4 allele had a shorter relapse-free time (p=0.023)
and a lower disease-free survival (p=0.012) compared with
women heterozygous or homozygous for the wild-type allele.
Overall survival was not statistically different (p=0.169).
Genotypes identified in DNA extracted from tumor samples
and buccal swabs were compared; the concordance between
buccal swabs and tumor samples was 100% for CYP2D6*4 (15
women) and CYP3A5*3 (13 women). These additional data
indicate that buccal swabs may be used in place of paraffinembedded tumor samples to predict clinical outcomes.
4.
Annotated Bibliography
1.
Pharmacogenomics
48
function of the dose. The doses were defined as low (less than
150 mg/m 2), medium (150250 mg/m 2), and high (more than
250 mg/m 2) based on the most common doses administered
to patients. Patients who were carriers of this variant allele
and received higher doses of irinotecan were more likely
to develop severe toxicity compared with patients with the
same genotype receiving lower doses. When patients with the
UGT1A1*28/*28 genotype were compared with patients who
were UGT1A1*1/*1 or UGT1A1*1/*28, the risk of toxicity in
the first group was greater at medium doses (odds ratio [OR]
= 3.22, 95% confidence interval [CI] = 1.526.81, p=0.008)
and high doses (OR = 27.8, 95% CI = 4195, p=0.005). At
lower doses, the risk was not statistically different (OR =
1.8, 95% CI = 0.378.84, p=0.41). This study suggests that
genotyping should only be conducted in patients receiving
higher doses (i.e., more than 150 mg/m 2) of irinotecan.
5.
6.
7.
9.
8.
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Pharmacogenomics
Pharmacogenomics
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Self-Assessment Questions
44. A 37-year-old African American woman with earlystage breast cancer recently completed adjuvant
chemotherapy after a lumpectomy. The woman is
premenopausal, and the tumor was hormone receptor
positive. Her medical history notes she does not respond
to codeine, and she experiences substantial adverse
effects after taking paroxetine for depression. Which
one of the following is the best endocrine therapy for
this woman at this time?
A. Oophorectomy.
B. Tamoxifen.
C. Anastrozole.
D. Goserelin.
Pharmacogenomics
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Pharmacogenomics
Pharmacogenomics
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