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Pharmacogenomics

Stacy S. Shord, Pharm.D., BCOP


Reviewed by Christine M. Walko, Pharm.D., BCOP; Katherine H. Chessman, Pharm.D., FCCP, BCPS, BCNSP; and Rodney
Gedey, Pharm.D., BCPS

Learning Objectives

single nucleotide polymorphism causes a change in the


amino acid and, ultimately, the protein. Other examples of
polymorphisms include copy number polymorphisms, in
which multiple copies of an entire gene exist; and insertions
or deletions, in which DNA bases are added or removed
compared with the wild-type DNA sequence.
Many studies now indicate that genetic information
may be predictive of response or tolerability to a specific
drug or provide prognostic information regarding the
disease. In the past few years, substantial progress has
been made in incorporating pharmacogenetics into clinical
practice. The inclusion of pharmacogenetic information
in package inserts, as well as the availability of U.S. Food
and Drug Administration (FDA)-approved genomic tests,
demonstrates the importance of this information.
These new developments have made genomic testing
feasible within the clinical arena, and some insurance
companies now reimburse the costs of many of these
tests. However, these tests are not being used widely. One
limitation to performing genetic tests is the ability to
interpret and make recommendations for therapy based on
the results; another is that most pharmacogenetic data are
from whites; thus, less is known about other ethnicities.
Still other limitations include variability between the sexes
and the lack of standardization of methods for identifying
specific genetic changes. For pharmacists involved in the
selection and recommendation of appropriate drug therapy,
a thorough understanding of pharmacogenomics and its
application to drug therapy is necessary. This chapter focuses
on areas of cancer therapeutics in which pharmacogenetic
information has been evaluated and has proved effective in
predicting clinical response or tolerability.

1. Distinguish between the different types of mutations


(e.g., single nucleotide polymorphisms, repeat
polymorphisms) associated with variations in
therapeutic response and toxicity in patients who
receive anti-cancer drugs.
2. Predict response and tolerability to anti-cancer drugs
based on genetic and nongenetic factors.
3. Interpret pharmacogenetic data with respect to selecting
appropriate anti-cancer drug therapy.
4. Modify the treatment regimen for a patient with cancer
using pharmacogenetic information.
5. Assess how the use of pharmacogenetic data can
improve therapeutic outcomes associated with anticancer drugs.

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
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Pharmacogenomics

subvariants, have been identified to date. Phenotype prediction


based on drug concentrations or genotype is common, and
more than one system is described. The phenotype can
be characterized as ultraextensive metabolizer, extensive
metabolizer, intermediate metabolizer, or poor metabolizer.
The ultraextensive metabolizers possess multiple copies
of functional alleles (e.g., *1, *2) and metabolize drugs
faster compared with extensive metabolizers. Intermediate
metabolizers typically possess one reduced function (e.g.,
*3, *4, *17, *29, *41) or null allele (e.g., *5), and poor
metabolizers typically possess two reduced function or
null alleles. These metabolizers break down drugs slower
when compared with extensive metabolizers. About 7%
to 10% of whites and 2% to 5% of African Americans
poorly metabolize CYP2D6 substrates. The most common
alleles associated with poor metabolic status in whites are
CYP2D6*3, *4, and *5, and in African Americans, they are
CYP2D6*17 and *29.
Two studies strongly indicate that variability in CYP2D6
activity or expression may affect the metabolism of tamoxifen.
The concentrations of tamoxifen and its metabolites were
measured in 12 women receiving tamoxifen therapy before
and after 4 weeks of paroxetine therapy. Paroxetine is a
selective serotonin reuptake inhibitor (SSRI) and a known
inhibitor of CYP2D6 that is commonly prescribed to
minimize hot flashes associated with tamoxifen. Endoxifen
concentrations decreased significantly (from 12.4 ng/mL
to 5.5 ng/mL) after paroxetine treatment. Furthermore,
endoxifen concentrations decreased less in women with a
variant CYP2D6 genotype (24%) compared with women
with a wild-type CYP2D6 genotype (64%).
In a follow-up study to validate these initial findings,
plasma concentrations of tamoxifen and its metabolites
were measured in 80 women 1 and 4 months after beginning
adjuvant tamoxifen therapy; the concentrations of the
metabolites were at steady state 4 months after starting
tamoxifen. The SSRIs included paroxetine, fluoxetine,
setraline, citalopram, and venlafaxine (in order of mostpotent to least-potent inhibitor of CYP2D6). Genotypic
analysis was performed for CYP2D6, CYP2C9, CYP3A5,
and SULT1A1. Mean endoxifen concentrations were 55%
lower in heterozygotes compared with homozygotes for
wild-type CYP2D6; the variant alleles examined included
*1 (wild-type) and *3 to *6. Furthermore, among patients
homozygous for wild-type genotype, mean plasma
endoxifen concentrations were 58% lower in patients taking
SSRIs compared with patients not taking SSRIs. Endoxifen
concentrations also were lower in women taking paroxetine
compared with those taking venlafaxine, consistent with
paroxetine being the most potent inhibitor and venlaxafine
being the least potent inhibitor of CYP2D6 when SSRIs are
compared. The mean plasma concentrations for endoxifen,
4-hydroxytamoxifen, and N-desmethyl-tamoxifen were
not associated with genotypes for CYP2C9, CYP3A5, or
SULT1A1. These studies demonstrate that the CYP2D6
phenotype and genotype are strongly associated with
endoxifen concentrations. However, it was not clear from
these studies whether these pharmacokinetic changes
influence clinical outcomes because response and
tolerability were not assessed. Later studies, as discussed
in the following section, suggest a possible relationship

Abbreviations in
This Chapter
ALL
AML
ATP
CML
CYP
DPD
EGFR
FISH
IHC
KRAS
SSRI
SULT
TPMT
UGT

Acute lymphoblastic leukemia


Acute myeloid leukemia
Adenosine triphosphate
Chronic myeloid leukemia
Cytochrome P450
Dihydropyrimidine dehydrogenase
Epidermal growth factor receptor
Fluorescence in situ hybridization
Immunohistochemistry
Kirsten retrovirusassociated
DNA sequences
Selective serotonin reuptake inhibitor
Sulfotransferase
Thiopurine methyltransferase
Uridine diphosphateglucuronosyltransferase

up to 15 years after discontinuing therapy. The aromatase


inhibitors (e.g., anastrozole, letrozole) are reasonable
alternatives to tamoxifen in postmenopausal women with
hormone receptorpositive breast cancer. Many studies
indicate that the aromatase inhibitors further decrease
recurrence, cause fewer adverse events than tamoxifen, and
may improve survival when administered sequentially with
tamoxifen. However, recent data supporting an association
between tamoxifen and metabolic status raise the question
whether the outcomes with tamoxifen may rival the outcomes
of aromatase inhibitors if the studies comparing these drugs
include only extensive metabolizers of tamoxifen.
Tamoxifen is a prodrug with a complex metabolic
profile; it undergoes extensive phase 1 metabolism by
several cytochrome P450 (CYP) enzymes, including
CYP3A4 and CYP2D6. Cytochrome P450 enzymes,
including CYP3A4, catalyze the metabolism of tamoxifen to
N-desmethyl-tamoxifen, which is subsequently metabolized
by CYP2D6 to endoxifen. The metabolism of tamoxifen
to 4-hydroxytamoxifen is also catalyzed by CYP2D6.
These metabolites can undergo additional metabolism
by sulfotransferase-1A1 (SULT1A1) and by uridine
diphosphate-glucuronosyltransferase (UGT), specifically
UGT2B7 or UGT2B15.
Tamoxifen and its metabolites, 4-hydroxytamoxifen
and endoxifen, can bind to estrogen receptors and
suppress estrogen-dependent cell proliferation. Both
metabolites demonstrate greater affinity for the estrogen
receptor compared with tamoxifen and greater potency in
inhibiting estrogen-dependent cell proliferation. Endoxifen
concentrations exceed those of 4-hydroxytamoxifen by 10fold, suggesting that endoxifen is the metabolite responsible
for the beneficial clinical outcomes associated with
tamoxifen.
CYP2D6 Polymorphisms
The CYP2D6 enzyme accounts for about 5% of total
hepatic CYP protein and metabolizes about 30% of drugs.
About 70 CYP2D6 variant alleles, together with additional
Pharmacogenomics

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Pharmacotherapy Self-Assessment Program, 6th Edition

between CYP2D6 and clinical outcomes in women receiving


adjuvant tamoxifen.

is associated with increased enzyme activity, whereas the


most common variant allele, SULT1A*2, is associated with a
2-fold reduced metabolic capacity compared with the wildtype allele. It was anticipated that these polymorphisms would
affect the therapeutic response associated with tamoxifen
because further metabolism of 4-hydroxytamoxifen would
terminate its effects on estrogen receptors.
Women taking tamoxifen and identified as homozygous
for SULT1A1*2 had a greater risk of death than women
identified as heterozygous or with homozygous wildtype genotype. Similarly, women identified as carriers
of UGT2B15 high-activity genotypes experienced more
disease recurrence and shorter survival times than those
without the genotype. Women homozygous for SULT1A1
trended toward improved disease-free recurrence compared
with women homozygous for the wild type, confirming
earlier findings. Furthermore, women homozygous for
the CYP3A5*3 allele who received tamoxifen for 5 years
had longer recurrence-free survival than those treated for
a shorter duration; however, this improvement was not
significant for women receiving tamoxifen for 2 years. These
retrospective analyses support an association between phase
2 metabolic status and outcomes of tamoxifen therapy, but
additional data are warranted to confirm these preliminary
findings, especially in light of recent data indicating that
endoxifen is a more potent active metabolite compared with
4-hydroxytamoxifen. Prospective studies in which patients
are treated based on genotype are warranted to confirm
these data before metabolic status for phase 2 enzymes can
be considered in treatment decisions.

Genotype and Clinical Outcomes


Using paraffin-embedded tissue blocks from 223
women with hormone receptorpositive breast cancer, the
association between the CYP2D6 genotype and tamoxifen
efficacy was evaluated. The DNA for CYP2D6*4, CYP2D6*6,
and CYP3A5*3 was amplified in at least 190 tissue blocks.
Women with the CYP2D6*4/*4 genotype had shorter relapsefree times and disease-free survival rates, but they did not
have significantly different overall survival rates. However,
more women with this genotype had lymph nodepositive
disease, which is a negative prognostic factor for women with
early-stage breast cancer. These women also experienced
less-severe hot flashes, an effect associated with tamoxifen.
No CYP2D6*6 alleles were detected, and CYP3A5*3
alleles were not associated with tamoxifen efficacy. In an
updated analysis, the investigators examined the medical
records to determine whether CYP2D6 inhibitors were
prescribed with tamoxifen. The women were characterized
as extensive metabolizers, intermediate metabolizers, or
poor metabolizers. Extensive metabolizers were women
without the CYP2D6*4 allele or coadministered a CYP2D6
inhibitor. Women with decreased metabolism, including
intermediate and poor metabolizers, experienced a shorter
time to recurrence and poorer relapse-free survival; poor
metabolizers demonstrated worse outcomes compared with
intermediate metabolizers. These initial findings suggest
that the changes observed in mean endoxifen concentrations
in association with the CYP2D6 genotype influence both
response and tolerability.
Four additional studies were published that examined the
CYP2D6 genotype as a marker of response to tamoxifen. The
first study enrolled 206 women receiving tamoxifen and 280
women not receiving therapy. Women heterozygous for the
CYP2D6*4, *5, *10, and *41 alleles and taking tamoxifen
had increased disease recurrence, shorter relapse-free
periods, and poorer event-free survival rates than women
not receiving therapy; these results support the data from
the first study described. Furthermore, a positive association
between the CYP2C19 genotype and tamoxifen outcomes was
found. Women heterozygous for CYP2C19*17 had a more
favorable outcome compared with women heterozygous for
*1, *2, and *3 alleles; of note, the *17 allele is associated
with increased catalytic activity. One retrospective analysis
supported an association between the CYP2D6 genotype
and higher disease-free survival rates, whereas two
retrospective studies did not find an association between
the CYP2D6 genotype and disease recurrence or survival in
women taking tamoxifen. Conflicting data are likely caused
by differences in study design and patient population and
should be confirmed prospectively. Additional limitations to
these studies are the small number of alleles evaluated and
the lack of consistent reporting of concomitant drugs that
may also affect endoxifen levels.
Associations between survival and genotype of other
phase 1 and two phase 2 metabolic enzymes in women
taking tamoxifen were also examined in some of these latter
studies. The enzymes UGT2B15 and SULT1A catalyze the
glucuronidation and sulfation of 4-hydroxytamoxifen,
respectively. The most common variant allele, UGT2B15*2,
Pharmacotherapy Self-Assessment Program, 6th Edition

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.
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Pharmacogenomics

Irinotecan

genotype and response rate was found. These studies


together support an association between UGT1A1*28 and
grade 3 or 4 toxicities; however, a relationship between
efficacy and genotype has not been clearly identified.
A recent meta-analysis suggested that the association
between the UGT1A1 genotype and grade 3 or 4 toxicity
may only be evident when a patient receives doses of
irinotecan greater than 150 mg/m2. This meta-analysis
included all of the individual studies described previously.
The analysis indicated that patients who were homozygous
for the UGT1A1*28 allele and who received doses greater
than 250 mg/m2 were more likely to develop hematologic
toxicity than patients receiving lower doses of irinotecan.
In contrast, patients homozygous for this variant allele who
received doses less than 150 mg/m 2 were not more likely
to develop hematologic toxicity. No association between
UGT1A1 genotype, dose, and diarrhea was observed.
These data suggest that the UGT1A1*28 genotype may only
be associated with hematologic toxicity at higher doses.
Therefore, genotyping may not be beneficial in predicting
the development of hematologic toxicity in patients receiving
lower weekly doses.

Irinotecan is a camptothecin analog that inhibits


topoisomerase-1. This anti-cancer drug, which is commonly
used to treat colon cancer in combination with fluorouracil
or cetuximab, may also be considered for other solid
tumors, including nonsmall cell lung cancer. The doselimiting adverse effects of irinotecan include diarrhea and
neutropenia.
Irinotecan undergoes metabolism by carboxylesterases
to its active metabolite SN-38. This metabolite undergoes
further metabolism by UGT to a more soluble and polar
metabolite SN-38G that facilitates its elimination in the bile
and urine. The main glucuronosyltransferase responsible
for the glucuronidation of SN-38 is UGT1A1. Several
polymorphisms are described for this enzyme, but the most
common polymorphism is UGT1A1*28. This variant allele
contains seven tandem repeats in the promoter region of
the gene compared with six tandem repeats in the wildtype allele UGT1A1*1. The promoter region of the gene is
the regulator that facilitates transcription. The UGT1A1*28
allele is associated with reduced gene expression and
catalytic activity in human liver tissue.

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.

UGT Polymorphisms and Toxicity


Many studies indicate that the incidence of grade 3 or
4 toxicity with irinotecan treatment is higher in carriers of
the UGT1A1*28 allele. In one study, 20 patients received
irinotecan 300 mg/m2 over 90 minutes every 3 weeks. The
allele frequencies were 0.375 and 0.625 for UGT1A1*28
and UGT1A1*1, respectively. Three patients identified as
homozygous or heterozygous for the variant allele developed
grade 3 or 4 neutropenia or diarrhea compared with no patient
identified as homozygous wild-type genotype. The more
severe toxicity was attributed to decreased glucuronidation
in the patients with the UGT1A1*28 allele.
In a larger study, the prevalence of grade 4 neutropenia
was 50% for patients homozygous for the variant allele,
12.5% for patients heterozygous for the variant allele,
and 0% for patients homozygous for the wild-type allele.
Furthermore, pretreatment bilirubin concentrations
correlated with the UGT1A1 genotype; because this enzyme
is responsible for the conjugation of bilirubin, these data
suggest that bilirubin may be used as a phenotypic marker
for genotype. Five additional studies confirm these data by
indicating that the variant allele is associated with greater
toxicityeither neutropenia or diarrhea. In contrast, two
studies indicate that the UGT1A1 genotype does not predict
toxicity. Different patient populations and chemotherapy
regimens may be responsible for the apparent differences in
outcomes, but the consensus appears to be that the UGT1A1
genotype is associated with toxicity.
Only one study to date has evaluated the association
between UGT1A1*28 and toxicity, response rate, and
overall survival. The incidence of neutropenia was 4-fold
higher in heterozygotes of UGT1A1*28, and the incidence of
neutropenia and diarrhea was 8.6-fold and 4.1-fold higher in
homozygotes of UGT1A1*28. A univariate analysis indicated
that carriers of this allele showed a trend toward poorer
overall survival; however, this association was not confirmed
as part of the multivariate analysis. No relationship between
Pharmacogenomics

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.
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Pharmacotherapy Self-Assessment Program, 6th Edition

One prospective study supports tailoring fluorouracil


therapy based on genetic factors. The study was conducted
to determine whether genetic factors would be helpful
in individualizing fluorouracil therapy. Toxicity was
linked to a poor performance status of the patient, a low
dihydrouracil-to-uracil ratio, and DYPD*2A in patients
receiving fluorouracil as first-line therapy for advanced
colorectal cancer. Prospective studies designed to tailor
therapy using these factors are needed before screening for
DPD deficiency is widely used in the clinical setting.

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.

DPD Expression and Efficacy


Dihydropyrimidine dehydrogenase expression may
be associated with the clinical response of solid tumors
to fluorouracil. Gene expression, protein expression, and
activity levels of DPD alone and in combination with
various enzymes involved in the metabolism and response
to fluorouracil have been examined in tissue blocks. Lower
intratumoral DPD expression or activity was associated with
improved response to fluorouracil in patients with head and
neck, breast, gastric, and colon cancers; however, the findings
vary from study to study and should be interpreted with
caution. Furthermore, relative gene or protein expression
does not appear to correspond with DPD activity, further
limiting the interpretation of these studies.
Clinical Testing
Screening for DPD deficiency includes both phenotypic
and genotypic methods. The most promising assay for
clinical use appears to be the dihydrouracil-to-uracil ratio
because this ratio corresponds to fluorouracil clearance,
and the assay has been streamlined to improve clinical
usefulness. This ratio also corresponds with fluorouracil
plasma concentrations and toxicity in patients receiving
adjuvant therapy for colorectal cancer.
Genotypic assays include measurement of DYPD
messenger RNA relative expression or copy number, but it
is not clear whether relative gene expression corresponds
with DPD enzymatic activity. These methods focus on
identifying the most common polymorphisms, but they
require improvement before clinical application can be
considered.
It is not yet possible to screen patients for DPD deficiency.
Testing methods will need to be validated in prospective
studies and demonstrate a positive impact on clinical
outcomes without a negative economic impact. Empiric
dosing, followed by dose modifications for myelosuppression
or hepatic impairment, remains the standard of care.

DPD Deficiency and Toxicity


Because more than 30% of patients receiving fluorouracil
describe severe toxicity, a causal relationship between DPD
deficiency and toxicity after fluorouracil has been examined
for about 2 decades. Reduced DPD activity is associated
with increased toxicity with fluorouracil and capecitabine
because of increased accumulation of the pyrimidine analogs.
Three cohort studies and five case reports demonstrated a
correlation between specific DYPD mutations and grade 3
or 4 toxicities after fluorouracil. The most common variant,
DYPD*2A, should not be singled out; one case series indicated
normal DPD activity was associated with lethal toxicity.
The DYPD*2A was found in only 2 of 93 patients and was
not associated with toxicity. Four additional studies found
that DPD deficiency was associated with increased toxicity
with fluorouracil or capecitabine. In these studies, DPD
deficiency was defined using plasma dihydrouracil-to-uracil
ratio alone or in combination with genotype. Of interest,
some patients included in these studies who experienced
severe toxicity were not carriers of the DYPD*2A allele.
Overall, ample evidence suggests a strong relationship
between fluorouracil exposure, DPD activity, and severe
hematologic and gastrointestinal toxicities.
Mild toxicities have been associated with high and low
DPD activity. Grade 1 or 2 neutropenia was associated with
elevated DPD activity in peripheral blood mononuclear
cells, whereas grade 1 or 2 dermatologic toxicity was
associated with reduced DPD activity. These data suggest
that tailoring fluorouracil therapy may be difficult because
different toxicities are associated with either high or low
DPD activity.
Pharmacotherapy Self-Assessment Program, 6th Edition

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
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Pharmacogenomics

that patients homozygous for a variant allele are more likely


to have poorer outcomes. Although response rates were not
examined in this study, the known relationship between
dose intensity and outcomes leads to the conclusion that
multiple dose interruptions that occur in patients with
TPMT deficiency receiving standard doses lead to poorer
outcomes. Therefore, these patients should receive lower
doses to permit the maintenance of dose intensity and
increase the likelihood of improved outcomes. The lower
dose also permits patients to receive full doses of other
myelosuppressive drugs.
Dosing adjustments before beginning therapy are
recommended for individuals with TPMT deficiency. For
intermediate metabolizers, the dose should be reduced to
65% of the standard dose; for poor metabolizers, the dose
should be reduced to 6% to 10% of the standard dose.
These adjustments are based on the reported percentage
of weeks that patients heterozygous or homozygous for a
variant allele were able to tolerate standard therapy with
mercaptopurine as part of maintenance therapy for ALL.
If an individual patient tolerates the dose, the dose can be
gradually increased to maximize response without causing
severe neutropenia.
Reports from small numbers of patients suggest a
relationship between TPMT activity and long-term adverse
effects. Thiopurine methyltransferase deficiency is associated
with an increased incidence of brain tumors and secondary
leukemias. In a study conducted at St. Jude Childrens
Research Hospital, the 8-year cumulative incidence of brain
tumors was 43% among children with TPMT deficiency
compared with 8.3% among children without TPMT
deficiency. Two studies indicate that TPMT activity might
be lower in patients who develop secondary acute myeloid
leukemia (AML) after receiving mercaptopurine for ALL. In
one study, 55 patients with lower TPMT activity (defined as
less than 14 units/mL of red blood cells) had a 5-year risk of
AML of 9% compared with 1% for the patients with higher
activity. The leukemogenic effect may also be increased in
individuals with low TPMT activity when mercaptopurine
is administered with other cytotoxic anti-cancer drugs.

produces an inactive metabolite, 6-methylmercaptopurine.


S-adenosylmethionine serves as the methyl donor of the
reaction. This enzyme also catabolizes mercaptopurine
nucleotides to inactive methylated nucleotides.
TMPT Polymorphisms
Thiopurine methyltransferase activity has large
variability in whites, with a trimodal distribution observed
in most individuals possessing high activity. About 10%
of individuals possess intermediate activity, and 1% have
low activity. These individuals may be characterized as
extensive, intermediate, or poor metabolizers. Extensive
metabolizers possess two wild-type alleles, intermediate
metabolizers possess one variant allele associated with
reduced metabolic capacity, and poor metabolizers possess
two variant alleles associated with reduced metabolic
capacity. The gene is inherited as an autosomal codominant
trait, and at least 19 variants have been identified to date.
The most common polymorphisms in whites are
TPMT*2 and TMPT*3A alleles, which account for up to
95% of reduced enzymatic activity. The frequencies of
mutations and the most common variant alleles differ by
ethnic population. For example, Indian Asians have a lower
frequency of variant TPMT alleles compared with whites,
and all variant alleles identified to date are TPMT*3A. In
comparison, African Americans have a frequency of variant
alleles similar to whites, but all variant alleles in identified
to date are TPMT*3C. The contribution of the remaining
variant alleles to reduced activity is not defined in whites or
other populations. Other variants identified include variable
numbers of tandem repeats in the 5-flanking promoter
region of the gene. The impact of these tandem repeats on
enzymatic activity is not clear; one study demonstrates
minimal declines in activity, whereas other studies
demonstrate no change in activity.
TMPT Deficiency and Clinical Outcomes
It is documented that reduced TPMT activity is associated
with an exaggerated or poor response dependent on genotype
response or severe hematologic toxicity. Individuals
heterozygous for one variant allele tend to have improved
response rates compared with individuals homozygous for
the wild type. Studies indicate that individuals labeled as
extensive metabolizers of standard doses of mercaptopurine
are at about a 3-fold greater risk of relapse compared with
intermediate metabolizers (heterozygotes) with standard
doses. It appears individuals identified as heterozygotes for
a variant TPMT allele may demonstrate improved outcomes
compared with homozygotes for the wild-type allele using
standard doses based on one study.
Individuals homozygous for a variant allele tend to
have a greater risk of severe myelosuppression when
receiving standard doses. The total population in one
study received 89% of the planned maintenance dose. The
planned maintenance dose did not differ between patients
identified as homozygous wild type or heterozygous for
a variant allele associated with reduced TPMT activity,
but the planned maintenance dose was reduced to 53%
secondary to myelosuppression in patients identified as
homozygous variant allele. About 12% of patients were
identified as carriers of a variant allele. Because dose
intensity is strongly associated with outcomes, it is possible
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

Pharmacotherapy Self-Assessment Program, 6th Edition

Transactivating mutations show a high concordance with


activity assays in patients with cancer and healthy volunteers,
suggesting genotype can be used in place of phenotype for
detecting TPMT deficiency. Genotype methods used to
detect the mutations include polymerase chain reaction,
followed by restriction fragment length polymorphism
or, more recently, pyrosequencing for the most common
mutations.
The product information for mercaptopurine summarizes
the data regarding the relationship between TPMT and
myelosuppression as described above. A statement included
in the product information supports TPMT testing when
patients experience severe myelosuppression after induction
chemotherapy. Furthermore, the product information
contains a statement that a patient with little or no TPMT
activity is at an increased risk of severe myelosuppression
and typically requires substantial dose reductions. No
specific dose recommendations are provided.
Overall, a strong association between low TPMT
activity and myelosuppression after mercaptopurine is
well documented. Limitations in TPMT testing include
lack of concordance between the various methods and lack
of validation in different ethnic populations. Thiopurine
methyltransferase testing in all patients is optimal, but the
testing is not routinely performed in all patients receiving
mercaptopurine for ALL. It is believed the relative cost and
low prevalence of TPMT deficiency is responsible for lack
of testing in all patients before beginning therapy. However,
TPMT testing should be strongly considered when a patient
develops severe myelosuppression, and the dose should
be adjusted to minimize dose reductions of concurrent
anti-cancer drugs and maintain thioguanine nucleotide
concentrations in agreement with the product information.

Cetuximab is used to treat both head and neck and


colorectal cancers. This antibody is also used to treat locally
or regionally advanced head and neck cancer in combination
with radiation therapy or recurrent disease alone. It is also
indicated for first-line therapy for patients with metastatic
colorectal cancer alone or in combination with irinotecan, as
well as recurrent disease. Panitumumab is also used to treat
patients with metastatic colorectal cancer whose previous
therapy with regimens containing fluoropyrimidines,
irinotecan, and oxaliplatin failed. Tumor regression and
disease stabilization occur in all of these settings.
EGFR Mutations and Efficacy
Mutations
Mutations in the EGFR coding and regulatory sequences
are reported. Most polymorphisms are considered activating
mutations and are associated with higher response rates
to these EGFR inhibitors. Often, these mutations are
associated with gene amplification. These mutations occur
in 15% of patients with a diagnosis of nonsmall cell lung
cancer who have a smoking history and in 50% of similar
patients without a smoking history. These mutations are
also common in squamous cell head and neck cancers with
EGFR protein identified in most tissue specimens. Other
tumors that may express these mutations include colon and
breast cancers and gliomas. Acquired resistance may be
associated with a secondary somatic mutation.
Erlotinib
Several clinical studies have documented a positive
association between EGFR somatic mutations in the kinase
domain (exons 1824) and clinical response to erlotinib. The
two most common mutations include an in-frame deletion
in exon 19 and a missense single-nucleotide polymorphism
L858R in exon 21, which account for 85% of the somatic
mutations identified in patients with nonsmall cell lung
cancer. These mutations are more common in patients with
clinical predictors of response to erlotinib (e.g., female sex,
Asian ethnicity, never-smoker, adenocarcinoma histology).
One study indicates that the patients receiving erlotinib or
gefitinib (another small molecule that can inhibit EGFR)
with an exon 19 somatic mutation experience longer overall
survival (38 months vs. 17 months) and higher response rate
(73% vs. 50%) compared with patients with the exon 21
missense mutation. Epidermal growth factor receptor copy
number or amplification is also associated with somatic
mutations and is positively associated with a clinical
response to erlotinib in several studies.
In a recent study, both Kirsten retrovirusassociated
DNA sequences (KRAS) wild-type and EGFR mutations
(in-frame exon 19 deletion and missense exon 21 mutation)
were associated with a positive response to erlotinib in
patients with nonsmall cell lung cancer. Significant
survival benefit from erlotinib therapy was observed for
patients with wild-type KRAS and EGFR positivity by
fluorescence in situ hybridization (FISH). Tumor specimens
were considered FISH positive if a high degree of polysomy
or amplification was noted; FISH permits semiquantification
of gene copy number. The KRAS gene encodes the protein
KRas, which promotes proliferation by stimulating
growth factors and other intracellular signaling pathways.
Kirsten retrovirusassociated DNA sequences are guanine

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

type KRAS are also more likely to respond to cetuximab


plus chemotherapy in chemotherapy-nave patients with
metastatic colorectal cancer. The response rate increased to
59% compared with 43% for chemotherapy alone, and the
risk of progression decreased to 25% compared with 43% at
1 year. It appears that EGFR gene copy number and KRAS
wild type are predictive of response to cetuximab in patients
with metastatic colorectal cancer.
Limited studies have examined the relationship between
these genes and response to cetuximab in head and neck
cancer. It appears that EGFR somatic mutations are very
common in this population, whereas KRAS mutations are
very rare. Therefore, identifying the subpopulations more
likely to respond to this antibody is unlikely, as indicated for
erlotinib.

triphosphatases downstream of EGFR. It is estimated that


KRAS mutations occur in 25% of patients with a diagnosis
of lung adenocarcinoma. Earlier studies also found that
KRAS mutations were predictive of resistance to erlotinib.
These mutations are also associated with poor prognosis
and response to cytotoxic chemotherapy. The KRAS
mutations can also aid in treatment selection in patients with
bronchoalveolar carcinoma or bronchoalveolar carcinoma
subtype.
These biomarkers have been studied extensively in
patients with nonsmall cell lung cancer, but not in patients
with other cancer diagnoses. However, erlotinib treatment is
being examined in clinical studies for the treatment of head
and neck cancers, glioma, and metastatic breast cancer. One
study using archived tissue indicated that EGFR expression
and localization could be associated with erlotinib response
in patients with squamous cell carcinoma of the head and
neck. Other studies appear to support the association between
high EGFR copy number in tumor specimens and response
to erlotinib in patients with head and neck cancer. Still other
studies appear to indicate that these mutations are rare and
not predictive of response. No distinguishing histology
or associated histories appear predictive of response in
patients with head and neck cancer; the disparate findings
are likely because of the high frequency of EGFR mutations
and KRAS wild type identified in tumor specimens of head
and neck cancers. Unlike the other tumors studied to date,
a specific mutation EGFRvIII appears responsible for the
noted responses to erlotinib in patients with gliomas. This
variant is a constitutively active genomic deletion unique
to glioblastomas and leads to the persistent activation
of the phosphatidylinositol 3 kinase (PI3K) signaling
pathway. One study demonstrated that coexpression of
this mutation and the phosphatase and tensin homolog
deleted in the chromosome 10 (PTEN) tumor-suppressor
protein corresponded to response to erlotinib in patients
with glioblastomas. It is postulated that the PTEN protein,
commonly lost in glioblastomas, maintains the link between
the EGFR and the PI3K pathway in this patient population.
It also appears that somatic mutations of the EGFR tyrosine
kinase domain may be predictive of response to this drug
in breast cancer. Erlotinib is not currently indicated for
breast cancer and gliomas, but it appears to be a promising
alternative therapy for some individuals with these unique
EFGR mutations.

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

Pharmacotherapy Self-Assessment Program, 6th Edition

derived growth factor receptor-beta. Both anti-cancer drugs


are indicated to treat patients with imatinib-resistant disease
and appear to demonstrate different sensitivities based on
the specific mutation identified as discussed below. Subtle
differences in the incidence of the more common adverse
effects attributed to these drugs may be caused by the
inherent differences in binding to the various receptor
tyrosine kinases.

survival benefit, EGFR status should be determined before


beginning therapy. In 2005, Genzyme launched the mutation
assay to detect EGFR mutations in patients with nonsmall
cell lung cancer. The assay combines polymerase chain
reaction and gene sequencing testing technologies to detect
EGFR mutations in the tyrosine kinase domain. The assay
is performed on tissue biopsy material in the laboratory by
trained scientists. Epidermal growth factor receptor testing
is considered standard of care.
In addition, KRAS testing is considered standard of care
in patients with colorectal cancer before they receive either
cetuximab or panitumumab. A KRAS mutation assay kit
that uses a polymerase chain reactionbased test to detect
the most common mutations is now available.

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.

Tyrosine Kinase Inhibitors


Chronic myeloid leukemia (CML) is characterized by
the translocation of chromosomes 9 and 22, known as the
Philadelphia chromosome. The fusion of these two genes
causes the formation of the BCR-ABL gene, which results in
an active tyrosine kinase protein. The phosphorylated protein
enhances cell proliferation, differentiation, adherence, and
apoptosis. Before the availability of imatinib, interferon
alfa and cytarabine was considered the standard of care.
Imatinib was one of the first tyrosine kinase inhibitors
developed; it works by inhibiting ATP binding to BCR-ABL
kinase, thereby blocking phosphorylation and downstream
signaling. The efficacy of imatinib in the treatment of
CML was first evaluated in a study in which 553 patients
were randomized to imatinib or interferon alfa plus lowdose cytarabine. Imatinib demonstrated superior efficacy
compared with interferon alfa and low-dose cytarabine in
rates of major cytogenetic responses, complete cytogenetic
responses, and rate of freedom from progression to the
accelerated phase and blast crisis. Imatinib became the
new standard of care for the treatment of CML based on its
greater efficacy and improved tolerability.
However, not all patients respond to therapy, and many
develop resistance. Primary resistance to imatinib is
defined as an initial lack of response to therapy; secondary
resistance occurs when patients achieve an initial response
but then develop progressive disease. Multiple mechanisms
have been proposed for the development of resistance to
imatinib. These include increased drug efflux by membrane
transporters, increased drug binding by plasma proteins,
constitutive activation of a downstream signaling pathway,
amplification of the BCR-ABL gene, and mutations within
the BCR-ABL kinase domain.
Two newly approved agents that target the BCR-ABL
fusion protein are nilotinib and dasatinib. These small
molecules were rationally designed based on known
mutations of the tyrosine kinase domain to overcome
resistance to imatinib. Nilotinib and dasatinib also bind to
the ATP-binding domain and inhibit BCR-ABL tyrosine
kinase activity. Nilotinib is a more potent BCR-ABL
inhibitor than imatinib, but it demonstrates activity similar
to other tyrosine kinases such as Kit and platelet-derived
growth factor receptor-beta. Dasatinib is also a potent
inhibitor of the BCR-ABL tyrosine kinase, as well as other
tyrosine kinases such as the Src family, Kit, and plateletPharmacotherapy Self-Assessment Program, 6th Edition

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

the newer tyrosine kinase inhibitors can elicit a response in


patients carrying the T315I mutation.
Omacetaxine, which is currently in clinical development,
may elicit a response in patients who carry the T315I
mutation. This anti-cancer drug has a novel mechanism of
action and induces apoptosis independent of tyrosine kinase
inhibition. Omacetaxine could provide a much-needed
effective therapy for carriers of the T315I mutation.

This review provides a thorough overview of the


polymorphisms in drug-metabolizing enzymes and
transporters that affect patients with cancer. Examples
include TPMT, DPD, and UGT. The authors discuss the
importance of associating changes in pharmacokinetics
with pharmacodynamics. Furthermore, the influence of the
underlying diagnosis and methodology for detecting these
polymorphisms are discussed. This review highlights the
need for prospective studies that show a clear relationship
between clinical outcomes and these polymorphisms before
clinical testing for these polymorphisms can be consistently
used to tailor treatment.

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.

2. Jin Y, Desta Z, Stearns V, Ward B, Ho H, Lee KH, et al.


CYP2D6 genotype, antidepressant use, and tamoxifen
metabolism during adjuvant breast cancer treatment. J Natl
Cancer Inst 2005;97:309.
This study was one of the first to evaluate the influence of
CYP2D6 and the use of concurrent CYP2D6 inhibitors on
the metabolism of tamoxifen. This study included 80 patients
with newly diagnosed breast cancer who were beginning
tamoxifen. Of these patients, 24 women were taking CYP2D6
inhibitors. Genotypes CYP2D6, CYP2C9, CYP3A5, and
SULT1A1 were assessed, and the concentrations of tamoxifen
and its metabolites were measured after 1 and 4 months of
therapy. Concentrations of endoxifen, the active metabolite of
tamoxifen, were significantly lower in women heterozygous
or homozygous for a variant CYP2D6 allele (p=0.003). In
patients who were homozygous wild-type genotype but
receiving CYP2D6 inhibitors, the plasma concentrations of
endoxifen were 58% lower than those who were not taking
CYP2D6 inhibitors (p=0.002). This study demonstrates a
definitive relationship between genotype and phenotype and
the influence of completive inhibitors on the disposition of
the active metabolite.

Conclusion

3.

The anti-cancer drugs discussed in this chapter


demonstrate substantial intersubject variability in response
to therapy. The variability may range from a poor response
to exaggerated toxicity in a subset of patients. It appears that
polymorphisms within the drug target (e.g., EGFR, BCRABL) or metabolic pathway (e.g., CYP2D6, TPMT, UGT,
DPD) may explain some of the variability. Studies show
an association between either efficacy or tolerability and
specific polymorphisms or expressions of these proteins.
Clinical testing is available for some of these mutations, but
an association between each known variant and outcomes
has not been identified. Furthermore, ethnic differences have
also not been explored for all of these mutations. However,
these tests may be helpful in reducing toxicity or maximizing
efficacy in some subsets and should be strongly considered
in these subsets. It is anticipated that clinical testing will
become more common, and that tailoring therapy based on
specific molecular characteristics will become the standard
of care.

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.

Hoskins JM, Goldberg RM, Qu P, Ibrahim JG, McLeod HL.


UGT1A1*28 genotype and irinotecan-induced neutropenia:
dose matters. J Natl Cancer Inst 2007;99:12905.
This meta-analysis included nine studies with a total of
821 patients and assessed the association between irinotecan
dose and severe hematologic toxicity in patients homozygous
for the variant allele UGT1A1*28. This study was the first to
demonstrate that toxicity associated with this variant allele is a

Annotated Bibliography
1.

Bosch TM. Pharmacogenomics of drug-metabolizing


enzymes and drug transporters in chemotherapy. Methods
Mol Biol 2008;448:6376.

Pharmacogenomics

48

Pharmacotherapy Self-Assessment Program, 6th Edition

maintenance therapy and demonstrated a trend toward


an association between genotype and dose interruptions.
The number of weeks of maintenance therapy omitted for
myelosuppression was similar between homozygotes and
heterozygotes for the wild-type genotype but markedly
lower for the patient identified as a homozygote for variant
genotype. This is one of the first studies to demonstrate the
impact of TPMT genotype on treatment.

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.

Yen JL, McLeod HL. Should DPD analysis be required prior


to prescribing fluoropyrimidines? Eur J Cancer 2007;43:1011
6.
This concise review discusses the metabolism of
fluorouracil and the relationship between DPD and clinical
outcomes. As discussed in the text, treatment of patients
with fluorouracil-based chemotherapy can be accompanied
by severe and sometimes lethal toxicity. Dihydropyrimidine
dehydrogenase plays a pivotal role in the metabolism of
fluorouracil, and DPD deficiency has been recognized as a
risk factor for the development of severe toxicity and clinical
outcomes. The various methods to measure DPD deficiency
are thoroughly discussed, including potential limitations
and eventual clinical application. Screening of patients for
the presence of a DPD deficiency before treatment is not
routinely performed, but the authors conclude that screening
for DPD deficiency may become standard of care as progress
continues regarding the understanding of the molecular basis
of the deficiency and the development of clinical assays.
Crews KR. Individualizing chemotherapeutic treatment of
colorectal cancer. Am J Health Syst Pharm 2006;63(Suppl
2):S12S17.
This review summarizes previously published reports
regarding polymorphisms in genes that encode drugmetabolizing enzymes, receptors of drugs used to treat
colorectal cancer, and the association between these
polymorphisms and clinical outcomes. The article focuses
on the relationship between fluorouracil and DPD deficiency,
irinotecan and UGT tandem repeats, and cetuximab and
EGFR mutations. The article also discusses tailoring
chemotherapy based on traditional risk factors in addition to
genetic polymorphisms. Assays used for genotypic testing,
protein expression, and metabolic capacity are described.
Overall, it provides a concise review of the data available for
patients with colorectal cancer and places the data in context
regarding clinical testing and therapeutic decisions.

Relling MV, Hancock ML, Rivera GK, Sandlund JT, Ribeiro


RC, Krynetski EY, et al. Mercaptopurine therapy intolerance
and heterozygosity at the TPMT gene locus. J Natl Cancer
Inst 1999;91:20018.
This article describes the first prospective study that
characterized the relationship between TPMT alleles and
deficiency. One hundred eighty patients received oral
mercaptopurine plus intravenous methotrexate as part of
the maintenance therapy for ALL. Thioguanine nucleotide
levels and TPMT activity were measured in red blood cells.
The activity of TPMT was used to identify the patients as
homozygous wild type, heterozygous, or homozygous variant.
The exact dose and reasons for deviations were recorded for
each dose. Mean thioguanine nucleotide values were inversely
related to TPMT activity (p<0.01); homozygous wild type
(n=161), 417 179; heterozygous (n=17), 963 752; and 3565
1282 homozygous mutant (n=2). Genotype was determined
in 28 patients and was in 100% concordance with phenotype.
These findings support the suggested dose reductions for
patients identified as carriers of a variant TPMT allele as
well as the current methods to identify TPMT deficiency by
genotype.

9.

Eberhard DA, Giaccone G, Johnson BE. Non-Small-Cell


Lung Cancer Working Group. Biomarkers of response to
epidermal growth factor receptor inhibitors in Non-SmallCell Lung Cancer Working Group: standardization for use in
the clinical trial setting. J Clin Oncol 2008;26:98394.
This article discusses in detail the options for assessing
the EGFR mutations in patients with nonsmall cell lung
cancer. The limitations of the current data are discussed and
illustrate the need for prospective trials to validate the current
assays. Furthermore, the need to standardize these assays is
discussed. Guidelines for tissue handling and analysis and
standardization of the current assays are provided. This
discussion is important to facilitate the understanding of
the discrepancies between the studies conducted to date and
the current limitations in determining the EGFR mutational
status before beginning therapy.

10. Zhu CQ, da Cunha Santos G, Ding K, Sakurada A, Cutz


JC, Liu N, et al. Role of KRAS and EGFR as biomarkers
of response to erlotinib in National Cancer Institute of
Canada Clinical Trials Group Study BR.21. J Clin Oncol
2008;26:426875.
Two major clinical studies demonstrated that EGFR
mutations predicted response to erlotinib and gefitinib. This
analysis evaluated the tissue of a subset of patients enrolled
into one of these landmark studies. Significant survival
benefit from erlotinib therapy was observed for patients
with wild-type KRAS (hazard ratio [HR]=0.69, 95% CI,
0.490.97, p=0.03) and EGFR FISH positivity (hazard ratio
[HR] = 0.43, 95% CI, 0.230.78, p=0.004) but not for patients
with mutant KRAS (HR = 1.67, 95% CI, 0.624.50, p=0.31),
wild-type EGFR (HR = 0.74, 95% CI, 0.521.05, p=0.09),
mutant EGFR (HR = 0.55, 95% CI, 0.230.78, p=0.12), or
EGFR FISH negativity (HR = 0.80, 95% CI, 0.491.29,

McLeod HL, Coulthard S, Thomas AE, Pritchard SC, King DJ,


Richards SM, et al. Analysis of thiopurine methyltransferase
variant alleles in childhood acute lymphoblastic leukaemia.
Br J Haematol 1999;105:696700.
The initial clinical studies that found an association
between TPMT deficiency and clinical outcomes in children
with ALL were conducted more than 10 years ago. This
study included 147 children with acute leukemia, and 11%
were identified as carriers of the most common variant allele.
Hematologic toxicity prevented patients from receiving
maintenance therapy on a mean of 11% of the weeks of

Pharmacotherapy Self-Assessment Program, 6th Edition

8.

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Pharmacogenomics

p=0.35). A multivariate analysis, however, indicated only


that EGFR FISHpositive status was prognostic for worse
survival in untreated patients (p=0.025) and predictive of a
greater survival benefit from erlotinib (p=0.005). This study
was one of the first to indicate that EGFR mutation status
alone may not be adequate to predict response to these small
molecules.
11. Baccarani M, Saglio G, Goldman J, Hochhaus A, Simonsson
B, Appelbaum F, et al. Evolving concepts in the management
of chronic myeloid leukemia: recommendations from an
expert panel on behalf of the European LeukemiaNet. Blood
2006;108:180920.
This article provides recommendations on the treatment
of patients with early chronic-phase CML. The most current
literature regarding the use of interferon alfa, transplantation,
and imatinib is reviewed. The use of imatinib and dosage
increases in patients who do not respond to therapy is also
discussed. In addition, the topic of resistance to therapy and
mutations is addressed. A list of the common mutations and
the IC50 (inhibitory concentration of 50%) values for imatinib
are provided. Definitions of response and monitoring, as well
as treatment recommendations, are provided. This review
article provides a concise overview of the treatment of CML
and a thorough description of the use of imatinib in patients
who develop resistance.
12. OHare T, Eide CA, Deininger MW. Bcr-Abl kinase domain
mutations, drug resistance, and the road to a cure for chronic
myeloid leukemia. Blood 2007;110:22429.
The authors review the most common mutations identified
in the BCR-ABL kinase domain and discuss how to treat
patients with these mutations. Several questions, including
how do point mutations cause resistance, when do mutations
occur, and what factors are driving resistance in patients, are
answered in this review. A colorful table identifies which
mutations demonstrate sensitivity to each of these small
molecules, and a discussion regarding the impact of nilotinib
and dasatinib on the emergence of these mutations is
addressed. This well-organized review helps readers address
concerns regarding the use of these inhibitors in patients with
CML.

Pharmacogenomics

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Pharmacotherapy Self-Assessment Program, 6th Edition

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.

41. A specific metabolic enzyme catalyzes the rate-limiting


step in the elimination of a new anti-cancer drug. The
maximal plasma concentrations of the parent drug vary
about 25-fold in patients. It is discovered that about
10% of patients develop substantial toxicity and can
no longer tolerate the drug. In this subset, parent drug
concentrations are significantly higher than anticipated.
No drug-drug interactions can be identified in these
patients. Molecular profiling indicates that messenger
RNA and concentrations of the metabolic enzyme are
substantially reduced. Furthermore, a base substitution
in the ligand-binding domain that leads to an amino acid
substitution is identified. Which one of the following
types of mutations is the most likely type of mutation
identified?
A. Single nucleotide polymorphism.
B. Tandem repeat.
C. Copy number.
D. Insertion.

45. A 53-year-old premenopausal woman recently given


a diagnosis of breast cancer just completed adjuvant
chemotherapy. She plans to take tamoxifen because her
tumor is hormone receptor positive. She is considered
a poor responder to codeine and hydrocodone, two
drugs that require metabolism by CYP2D6 to the active
compound. Which one of the following outcomes is
most likely based on her suspected CYP2D6 metabolic
status?
A. Increased disease-free survival.
B. Increased hot flashes.
C. Decreased overall survival.
D. Decreased relapse-free survival.

42. A mutation in a ligand-binding domain of a drugmetabolizing enzyme corresponds to a decrease in


the active metabolite and corresponding increases
in the parent drug in an animal model for a new
anti-cancer drug. Clinical studies subsequently
confirmed the relationship between the mutation and
the pharmacokinetics in humans, but Phase II studies
did not identify an association between the changes
in pharmacokinetics and increases in the incidence of
grade 3 or 4 toxicity or response. Genetic testing in
these studies included a relatively simple polymerase
chain reaction, followed by biallelic determination,
which may be performed in most laboratories. However,
this mutation occurs in only about 1% of whites, and
its frequency in African Americans, Hispanics, and
Asians is not known yet. Which one of the following
characteristics of this mutation is the best support for
clinical testing?
A. Simple, available assay.
B. Associated with toxicity.
C. Ethnic variability.
D. High prevalence.

46. A 65-year-old white man with a diagnosis of locally


advanced colon cancer received combination
chemotherapy with oxaliplatin and fluorouracil, but his
disease progressed after three cycles. Subsequently,
he will receive combination chemotherapy with
fluorouracil, irinotecan, and leucovorin every 2 weeks.
His laboratory values indicate he is unable to conjugate
bilirubin effectively. Which one of the following is the
most important action before beginning therapy with
irinotecan?
A. Provide filgrastim to minimize myelosuppression.
B. Start loperamide to minimize diarrhea.
C. Complete clinical testing for uridine diphosphateglucuronosyltransferase (UGT)1A1*28.
D. Reduce the irinotecan dose to 80% of the usual
starting dose.

43. A 45-year-old white woman recently completed adjuvant


chemotherapy for early-stage breast cancer. She is
premenopausal, and the tumor is hormone receptor
positive. Tamoxifen 20 mg/day given orally was initiated
immediately after the completion of chemotherapy. She
started taking paroxetine for depression. Within 1 year,
the breast cancer recurs. Which one of the following is
the most plausible explanation for her recurrence?
A. A drug-drug interaction.
B. She is a cytochrome P450 (CYP) 2D6 extensive
metabolizer.
C. Nonadherence.
D. Premenopausal status.
Pharmacotherapy Self-Assessment Program, 6th Edition

47. A 52-year-old Hispanic woman undergoes clinical


testing for UGT1A1 polymorphism after developing
severe neutropenia (absolute neutrophil count less than
500 cells/mm3) after receiving one dose of irinotecan.
The woman is receiving irinotecan in combination
with fluorouracil for first-line treatment of metastatic
colorectal cancer. The test indicates the woman is
homozygous for the variant allele. Which one of the
following alternatives is best for this woman at this
time?
A. Discontinue irinotecan; begin alternative therapy.
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Pharmacogenomics

B. Decrease the irinotecan dose to 20% of the usual


dose.
C. Administer filgrastim to increase white blood cell
count.
D. Reinitiate irinotecan at the current dose once the
white blood cell count increases.

food. Which one of the following is the most likely


explanation for this toxicity?
A. DPD deficiency.
B. Decreased carboxylesterase activity.
C. Drug-food interaction.
D. Drug-drug interaction.

48. A 50-year-old white man with a diagnosis of


metastatic colorectal cancer recently underwent
first-line chemotherapy, which failed. He is to begin
second-line therapy with fluorouracil, leucovorin, and
irinotecan every 2 weeks. The patient expresses normal
dihydropyrimidine dehydrogenase (DPD) activity
and is a carrier for the variant UGT1A1*28 allele.
Which one of the following most accurately describes
the potential impact of his metabolic capacity on his
response to or tolerance of this combination anti-cancer
drug regimen?
A. More severe bone marrow suppression with
fluorouracil.
B. Improved clinical response to fluorouracil.
C. More severe diarrhea and myelosuppression with
irinotecan.
D. A good response to irinotecan with prolonged
exposure to SN-38.

52. A 3-year-old Asian Indian boy with acute lymphoblastic


leukemia (ALL) completed induction and consolidation
therapy with myelosuppressive chemotherapy and
achieved complete remission. The boy then began
maintenance therapy with mercaptopurine, vincristine,
methotrexate, and prednisone. After one cycle, he
developed grade 3 neutropenia. Which one of the
following statements is the best reason to determine
thiopurine methyltransferase (TPMT) genotype in this
patient?
A. Severe myelosuppression developed with the first
cycle of chemotherapy.
B. Dose interruptions correspond with poorer
response.
C. TPMT mutations have not been identified in Asian
Indians.
D. Doses of other myelosuppressive drugs may be
modified.

49. A patient developed grade 3 or 4 neutropenia and


diarrhea after receiving a single irinotecan dose of 300
mg/m2 over 90 minutes. The patient is determined not
to be a carrier of the variant allele UGT1A1*28 after a
commercial assay is completed to identify the genotype
for this enzyme. Serum creatinine is 1.1 mg/dL, total
bilirubin is 1.3 mg/dL, and transaminases are 1.2 times
the upper limit of normal. Which one of the following
is the most plausible explanation for the development of
severe toxicity in this patient?
A. Reduced hepatic clearance of irinotecan.
B. Reduced urinary clearance of irinotecan.
C. Alternative mutations in the UGT1A1 gene.
D. Decreased carboxylesterase activity.

53. A 7-year-old girl with ALL is starting maintenance


chemotherapy with oral methotrexate 20 mg/m 2/day
and oral mercaptopurine 75 mg/m 2/day. She is 122
cm (48 inches) tall and weighs 11 kg (25 lb). Clinical
testing for TPMT shows that she is a heterozygous for a
variant allele associated with reduced metabolism and
increased grade 3 and 4 myelosuppression. Which one
of the following mercaptopurine doses is best for this
girl at this time?
A. 100 mg.
B. 65 mg.
C. 25 mg
D. 10 mg.
54. A 4-year-old boy with a diagnosis of ALL is prescribed
a complex chemotherapy regimen that includes
maintenance therapy with mercaptopurine. Basing
chemotherapy decisions on TPMT genotype is most
likely to result in which one of the following?
A. Reduced risk of drug-induced neutropenia.
B. Shortened course of chemotherapy.
C. Elimination of the need for hematologic
monitoring.
D. Reduced need for a multiple-drug chemotherapy
regimen.

50. A 68-year-old white man was given a diagnosis of


locally advanced pancreatic cancer. When he did not
respond to three cycles of gemcitabine, he opted to
start bolus fluorouracil with radiation therapy. If it were
available, which one of the following options would be
the best reason for offering clinical testing of DPD in
this man?
A. DPD activity is lower in African Americans.
B. DPD activity is lower in women than men.
C. DPD deficiency corresponds with increased
toxicity.
D. DPD deficiency corresponds with improved
outcomes.

Questions 55 and 56 pertain to the following case.


D.B. is a 70-year-old white man with a recent diagnosis
of Philadelphia chromosomepositive chronic myeloid
leukemia in the chronic phase. He is not a candidate for a
hematopoietic stem cell transplantation because of advanced
age. The patient was initiated on imatinib 400 mg/day given
orally. After 3 months, the patient achieved a hematologic
response, and at 6 months, the patient achieved a cytogenetic

51. A 63-year-old Hispanic woman begins capecitabine


plus lapatinib for metastatic breast cancer that had
progressed on previous systemic therapy. The woman
develops grade 3 neutropenia after starting therapy.
She takes capecitabine with food and lapatinib without
Pharmacogenomics

52

Pharmacotherapy Self-Assessment Program, 6th Edition

60. A 55-year-old man receives a diagnosis of recurrent


colorectal cancer after receiving oxaliplatin with
fluorouracil as first-line therapy and irinotecan plus
fluorouracil as second-line therapy. He will begin
taking cetuximab alone. Which one of the following
assays should be conducted as part of standard of care
before beginning therapy?
A. KRAS.
B. EGFR.
C. UGT.
D. DPD.

response. However, a bone marrow biopsy at 12 months


showed accelerated disease.
55. Which one of the following factors places D.B. at
increased risk of a tyrosine kinase domain mutation?
A. Age.
B. Race.
C. Disease phase.
D. Prior therapy.
56. A mutational analysis demonstrated that D.B. has a
T315I mutation. Which one of the following is the best
recommendation for therapy for D.B. at this time?
A. Imatinib 600 mg/day orally.
B. Dasatinib 100 mg/day orally.
C. Nilotinib 200 mg/day orally.
D. Participation in a clinical trial.
57. A 72-year-old white woman received a diagnosis of
stage IIIB adenocarcinoma of the lung. She also has a
104 pack-year history of smoking. Chemotherapy with
paclitaxel plus carboplatin was administered for four
cycles, but the patient experienced disease progression.
She plans to begin therapy with erlotinib. Which one
of the following patient and/or tumor characteristics is
most likely to be associated with an increased response
to erlotinib in this patient?
A. Smoking history.
B. Tumor subtype.
C. Age.
D. Race.
58. A 55-year-old Asian woman has a diagnosis of large
cell lung cancer. Her medical history includes no
illicit drugs or alcohol abuse, but she smoked about
one-half pack of cigarettes per day for 30 years. She
quit smoking about 7 years ago. Mutational analysis
indicates epidermal growth factor receptor (EGFR)
overexpression and Kirsten retrovirusassociated DNA
sequences (KRAS) variant. Which one of the following
characteristics is associated with a greater likelihood of
response to erlotinib in this woman?
A. Race.
B. Sex.
C. Tumor subtype.
D. KRAS mutation.
59. The mutation analysis for EGFR domain has been
examined in multiple studies. The tumor tissue from an
individual patient shows immunohistochemistry (IHC)
positive, fluorescence in situ hybridization (FISH)
negative, and EGFR altered sequence. Which one of
the following tests is the best predictor of response to
erlotinib?
A. Western blot.
B. IHC.
C. FISH.
D. Mutation analysis.

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