Chapter 1

Polymorphisms in Pharmacogenetics of Personalized

Cancer Therapy

Gizem Calibasi Kocal and Yasemin Baskin

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69207

Abstract

Therapy process of personalized cancer management covers surgery, chemotherapy,

radiation therapy and targeted therapies. The choice of cancer chemotherapeutic agents
and doses depends upon the location and stage of tumor, as well as the general state of
the patient. On the chemotherapy, radiotherapy, and targeted therapy processes, phar-
macogenetics offers customized solutions according to the personal genetic information.
Especially for clinicians, genetic information obtained from polymorphism-based phar-
macogenetic tests is highly crucial for the better prediction ability of drug response and
life-threatening toxic reactions due to the narrow therapeutic index of cancer chemo-
therapeutic agents. Pharmacogenotyping utilizes different examination strategies, such
as single nucleotide polymorphism analysis, somatic/germline mutation analysis and
partial/full genome sequencing. The promising effect of pharmacogenetics on the solving
of the individual variability in drug response and toxic reactions is being observed with
the accumulation of the information that unravel the human genomic variations from
large-scale population and multi-parameter-based pharmacogenetic studies of the post-
genomic era. Polymorphisms contribute wide variations in human genome and may
define how individuals respond to medications, either by changing the pharmacokinet-
ics and pharmacodynamics of drugs or by altering the cellular response to therapeutic
agents. To define the effect of polymorphisms on the targets of chemotherapeutics is
necessary for the prediction of altered pharmacokinetics of therapeutic agents.

Keywords: personalized medicine, pharmacogenetics, polymorphisms

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
4 Genetic Polymorphisms

1. Introduction

1.1. Genetic polymorphisms

1.1.1. Mutation or polymorphism

Genetic and environmental factors are the two main reasons that cause human phenotype
variations. If the genomic DNA sequences of two individuals are compared, substantial
sequence variations can be detected at different points of the whole genome. There are many
forms of these genetic variations [1]. Polymorphism term, arose from the combination of the
Greek words ‘poly’ (meaning as multiple) and ‘morph’ (meaning as form), is used in genet-
ics to describe multiple forms of a single gene that exist in a population. Polymorphisms are
genetic variants and refer to the occurrence of various phenotypes in a certain population.
A polymorphism is a DNA sequence variation and does not classify as mutation. In genetic
polymorphisms, there are two or more equally acceptable sequence of a gene and the com-
mon allele must have a frequency of 1% or more in the population. If the frequency is lower
than 1%, the allele is accepted as a mutation. On the other hand, a mutation is a change in
DNA sequence away from normal allele and forms abnormal variant [2].

1.1.2. Nomenclature

The unique and universal nomenclature to refer specific single nucleotide polymorphisms
(SNPs) is that using the rs number (reference sequence). It stands for Reference SNP clus-
ter ID. The rs number allows the precise identification of a polymorphic variation in the
numerous databases (NCBI, HapMap, SNP500 Cancer, etc.). For instance, a SNP causes a
replacement of an amino acid by another amino acid; this can be defined by the name and
the position of the replaced amino acid, followed by the name of the novel amino acid.
As an example, a ­common SNP in the DPYD gene is identified as rs 1801160 [V732I or
Val732Ile]. SNPs are also identified by the name and position of nucleotide in the reference
DNA sequence. The same SNP in the DPYD gene, presented with rs 1801160, is identified as
2194G>A. The letters A, T, C, and G can be used for both nucleotides and amino acids, and
this can cause confusion [3].

1.1.3. Types of polymorphisms

Developments in next generation sequencing technologies under the “Human Genome

Project,” simplify to investigate the allelic variants of a gene taken from different people
of a population [4]. These various genetic polymorphisms include minor changes on DNA
sequence, as substitutions, deletions, insertions, and repeats. These changes influence the
three-dimensional structure, expression and activity of the proteins encoded by these genes.
Alterations called as single nucleotide polymorphisms (SNPs, pronounced snip) are the most
common form of polymorphisms on a gene sequence. They occur when alleles reveal only a
single base pair (bp) change—A, T, C or G—in the genome sequence between individuals.
For example, one variant may have an A nucleotide at a certain position and other has a G
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nucleotide (Figure 1). This type of change generates single nucleotide variants (SNVs). SNPs
can affect gene function due to the change of protein but can also occur in noncoding parts of
the gene so they would not be seen in the protein product [5, 6].

1.1.4. Polymorphisms and ethnicity

Intrinsic factors and extrinsic factors cause variability in drug response. Extrinsic factors such as
food and concomitant medications may be controllable, but intrinsic factors such as gender, eth-
nicity, age, renal or hepatic function, and genetic differences in the expression of enzymes need
advance knowledge to control [7]. Ethnicity is one of the key factors that can explain the observed
variability in both pharmacokinetics (PK) and pharmacodynamics (PD) of therapeutics, result-
ing in differences in response to drug therapy as well as chemotherapeutics. United States Food
and Drug Administration (FDA) guidelines pay attention to “ethnically sensitive situations”
for some drugs and suggests the types of solutions that may control such ethnic sensitivity.
One of the most important factors that may contribute to this ethnic sensitivity of a drug, include
genetic polymorphisms in metabolic pathways of drugs [7, 8]. To define the variability on the
response or metabolism of specific therapeutics or drug targets, drug companies and presti-
gious research groups are biobanking DNA samples [9]. Although the clinical relevance of some
variants is well characterized, the relevance of some variant alleles is as yet unknown.

1.1.5. Allele frequency and Hardy-Weinberg principle

There are some challenges on the use of disease-associated polymorphism knowledge. One
of these challenges is the lack of the unique information regarding the frequency of spe-
cific polymorphism in the targeted population. Without a unique presenting style of the

Figure 1. Classes of DNA variation affecting a single nucleotide position. (A) Single nucleotide variant (SNV) in which
two variants differ by having a G nucleotide or a C nucleotide (B) Insertion variation in which variant 1 has exactly
same reference sequence, variant 2 has one more T nucleotide; Deletion variation in which variant 1 has a A nucleotide,
variant 2 does not have; (C) Variable number of tandem repeats (VNTR) in which two variants differ by having repeats
of nucleotides.
6 Genetic Polymorphisms

­ olymorphism-related data, prevention of the risk for disease and drugs remain unknown.
p
In addition, determining the factors that may affect the association of the allele with disease
or drugs, such as ethnicity, may not be possible without population-based allele frequencies
[10–12]. SNPs can be assigned with an allele frequency—the ratio of chromosomes in the pop-
ulation carrying the less common variant to those with the more common variant. It is impor-
tant to note that there are variations among different populations, so a common SNP allele may
be much rare in one geographical or ethnic group than another [13, 14]. The Hardy-Weinberg
principle can be used to calculate allele frequencies [15]. The Hardy-Weinberg principle (also
known as the Hardy-Weinberg equilibrium, equation, theorem, or law) states that allele fre-
quencies in a population will remain stable from generation to generation in the absence of
other evolutionary factors, such as mutation, polymorphisms, genetic drift, gene flow, meiotic
drive, and mate choice. Hardy-Weinberg principle describes that the ideal condition against
the effects of these factors can be analyzed. The principle is named after Godfrey Harold
Hardy and Wilhem Weinberg, who first demonstrated it mathematically. If Hardy-Weinberg
principle is violated, the key interferences of a genetic polymorphisms-based study may be
compromised. Thus, accumulating evidence suggests that Hardy-Weinberg principle-based
reporting may be optimal in genetic and nongenetic journals, because variability in the ana-
lyzed data can cause errors or peculiarities [15].

2. Polymorphisms-based pharmacogenetics applications and

personalized cancer therapy

Most of the chemotherapeutic agents for cancer treatment affect a minority of cancer patients
and have a narrow therapeutic index that frequently causes life-threatening toxicities and
even death. Even specific molecule-targeted therapies, which are safer than cytotoxic drugs,
are associated with severe adverse events. Thus, novel treatment strategies that can increase
the effectiveness of therapy and decrease the rate of adverse events will be developed. Under
this approach, the aim of personalized medicine is to tailor the therapy options according to
patient’s molecular profile [16, 17]. Establishing the relation between molecular characteris-
tics of patient and drug outcomes is crucial for the identification of predictive biomarkers and
understands the base of personalized therapy. Personalized medicine can also be called as P4
medicine due to the various contents as predictive, personalized, preventive, and participa-
tory medicine (P4); it separates patients into different groups with an individual’s molecular
profile. Thus, personalized medicine covers the determination of the safest and most effec-
tive chemotherapeutic agents [18]. The goal of discipline of pharmacogenetics, first used in
the late 1950s, is to make ‘personalized medicine’ as applicable to various patient groups.
It can be defined as the study of patients’ genotype affecting drug response. In some patient
groups, certain drugs work well but not as well in others. Pharmacogenetics-based studies
(between genotype of patients and the response of therapeutics) allow designing more effec-
tive and population-specific therapeutic treatments (Figure 2). Polymorphism analysis, muta-
tion analysis and genome sequencing are the backbones of discipline of pharmacogenetics.
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Figure 2. The traditional prescribing approaches in clinical therapies, such as ‘trial and error’, ‘one drug fits all’ and ‘one
dose fits all’, have limits due to the drug safety. Pharmacogenetics combines standard biochemical tests with molecular
genetics based tests to detect DNA variations in the human genome for the application of genomic-based prescribing.

Polymorphisms and their association with ­diseases should be handled based on gene as bio-
markers, due to the relatively large frequency in the human genome. To learn, how to use and
interpret the polymorphisms analysis and which test(s) should be chosen is essential.

2.1. Methods for polymorphism detection

After the deep sequencing of human genome with Human Genome Project, the detection
of population-based DNA polymorphisms, especially that effect the development and the
progress of diseases, is the second phase of human genomics. High-throughput polymor-
phism genotyping process includes fast and cost-effective identification of polymorphisms
in different individuals and lead to the determination of associations between genotype and
phenotype. Generally, genotyping steps start with the isolation of starting material as DNA
from patient; it follows with amplification to increase the sample amount and then finalize
with polymerase chain reaction (PCR), sequencing or array-based technologies. A number of
good polymorphism genotyping technologies are currently in use to meet the needs of clinics
and researches, but only one genotyping method is not ideal for all applications (Table 1).
Slow speed of assays due to the time-consuming protocols, high instrument and consumable
costs, and requirements on the performing multiple assays in parallel are the main challenges
of polymorphism genotyping technologies. Studies on ideal polymorphism genotyping tech-
nologies are on development process [19–21].
8 Genetic Polymorphisms

Method Advantages Disadvantages

Allele-specific single-base High success rate; High instrument cost;
primer extension High accuracy; Relatively long protocol;
Quantitative; Tricky multiplex PCR.
Adaptation to multiplexing.

Allele-specific enzymatic High accuracy; High instrument cost;

cleavage – restriction fragment SNP at a restriction endonuclease Long protocol;
length polymorphism (RFLP) recognition sequence. Difficult adaptation for high-throughput
genotyping

Pyrosequencing Provides sequence information High instrument and consumable cost;

surrounding the polymorphism; Long protocol;
Real-time scoring; Limited throughput
Quantitative.

Mass spectrometry High accuracy (as long as the sample is High instrument and consumable cost;
very pure); Long protocol;
Fast genotyping reactions in seconds; Limited throughput;
Allows thousands of reactions in a day. Requires very pure sample for accuracy.

Invader assay No PCR for amplification; Requires large amount of genomic DNA;
Simple protocol; Requires optimizations for each SNP;
Isothermal reaction Key enzyme is not suitable for general
research use.

Table 1. A summary of popular polymorphism detection methods with their advantages and disadvantages [19–21].

2.2. Polymorphisms as biomarkers for drug response and toxicity

Variations in the metabolism of a chemotherapeutic agent due to genetic alterations

may cause significant differences in terms of efficacy and toxicity. Such pharmacological
effects occur since oncologists schedule the dosing of chemotherapeutic agents accord-
ing to patient’s body surface area and other nongenetic factors. Genetic differences due to
the polymorphisms are thought as one of the strongest reasons in adverse drug reactions
(ADRs). Genetic polymorphisms are considered as molecular biomarkers in pharmacoge-
netic-based studies both in clinic and research to predict the ADRs and apply the medica-
tions as personal. The main objective of pharmacogenetics is to understand the nature of
various responses including adverse drug reactions (ADRs) to drugs [22]. Pharmacogenetic
associations are important in cancer chemotherapy due to the extremely narrow chemo-
therapeutic index of anticancer drugs given for cancer management. Polymorphisms in
both patient’s genome and tumor genome affect the regulation of drug transport, reten-
tion and efflux of anticancer drugs, determining the penetration into tumor tissue. Genetic
information of tumors is not stabile as somatic tissues; new alterations on genetic mate-
rial (as mutation or chromosomal loss) can occur continuously. Therefore, drug-related
toxicities depend on the genotype of nontumor tissue. Thus, the tumor genome possesses
most of the polymorphisms that influence the sensitivity or resistance of drugs (KRAS and
EGFR, KIT, TS polymorphisms, etc.); hence, treatment efficacy and tumor genome will have
a key role as a dose limiting factors in cancer management. Polymorphisms on the host
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genome, which tumor genome does not present, are the main determinants of toxicity risk
(e.g. ­polymorphisms on the genes of drug ­metabolism such as dihydropyrimidine ­dehydrogenase
gene (DPYD), ­thiopurine-S-methyltransferase (TPMT), UDP-glucuronosyltransferase (UGT), etc.)
[23]. Genotyping studies have revealed that the gene encoding a specific protein can have a
number of differences in sequence at the nucleotide level. These differences especially called
as polymorphisms, sometimes do not cause significant alterations on the final product, but
may have an effect on the substrate specificity and activity of the product (especially for
enzymes) or other characteristics and functions. For example, polymorphisms in cytochrome
P450 2D6 (CYP2D6) are one of the cytochrome P450 enzymes of the liver that can influ-
ence how humans metabolize cancer drugs, although the enzymes are basically the same
sequence and structure. Polymorphisms in CYP2D6 have been seen in the general popula-
tion about 10% and it has been associated with poor-metabolizer phenotype of enzyme.
This is important for codeine-based pain medications due to the activation of codeine to
morphine and includes CYP2D6-dependent step [24].

Pharmacogenetic tests based on the determination of genetic variants for drug efficacy or
toxicity has begun to use in the 2000s, although genetic-based studies began in the 1950s. FDA
has been working to improve pharmacogenetic technologies in the development, regulation
and use of medications and revised drug labels in terms of pharmacogenetic biomarkers in
oncology area (Table 2) [25, 26].
It is important to describe important gene polymorphisms and their clinical meaning in
oncology field that may determine the optimum pharmacological treatment in terms of
treatment outcomes, tolerability and the occurrence of serious, even life-threatening adverse
reactions.

Pharmacogenetic biomarker Drugs Labeling Outcome

UGT1A1 Irinotecan Dose determination and administration; Toxicity
Belinostat Warnings and precautions, clinical pharmacology
Nilotinib
Pazopanib

DPYD Capecitabine Warnings and precautions, patient counseling Toxicity

Fluorouracil information

TPMT Cisplatin Adverse reactions Toxicity

Mercaptopurine Dose determination and administration; Toxicity

Thioguanine Warnings and precautions; Adverse reactions;
Clinical pharmacology

G6PD Rasburicase Warnings and precautions; Contraindications; Toxicity

Dabrafenib Adverse reactions;
Patient counseling information

CYP2D6 Tamoxifen Dose determination and administration; Toxicity

Rucaparib Clinical pharmacology

Table 2. FDA-approved pharmacogenetic biomarkers for anti-cancer drug labeling [26].

10 Genetic Polymorphisms

2.2.1. Uridinediphosphate glucuronosyl transferase 1A1 (UGT1A1)

The UGT super family includes four main UGT families, namely UGT1, UGT2, UGT3, and
UGT8. The UGT1 and UGT2 genes, encode 16 functional proteins, have been extensively
studied and well characterized. A phase II metabolic enzyme, UGT1A1 is the most studied
UGT enzyme due to its main role in glucuronidation of exogenous and endogenous sub-
strates, including bilirubin. UGT1A1 also appears in the metabolism processes of most of the
anti-cancer drugs, such as topoisomerase I inhibitor irinotecan, the topoisomerase II inhibi-
tor etoposide [27]. Alterations on the glucuronidation activity of UGT1A1 caused by genetic
or environmental factors may have significant physiological and pharmacological results
on the metabolism of anticancer agents. Allelic variations have been identified in the pro-
moter region and exon 5 of UGT1A1 region. The wild-type allele of UGT1A1 gene (known
as UGT1A1*1) has six thymine- adenine (TA) repeats in the promoter region of gene (TATA
box). Allelic differences vary from five (UGT1A1*36, proficient allele) to eight (UGT1A1*37,
­deficient allele) TA repeats, and these differences affect the UGT1A1-mediated glucuronida-
tion of SN-38 (7-ethyl-10-hydroxycamptothecin), active metabolite of anticancer drug irino-
tecan both in vitro and in vivo. Increasing number of TA repeats has been associated with
decreased transcription of gene and overall UGT1A1 activity (Table 3) [28]. Patients (allele
frequency in Caucasians 8–20% are homozygous, 40–50% are heterozygous) with seven TA
repeat sequence (named UGT1A1*28) have severe toxicity risk after irinotecan treatment
because of decreased gene expression and overall UGT1A1 activity (30% enzyme activity in
*28 relative to *1 allele). These patients [with (TA)7 repeats] have fourfold relative toxicity risk
compared with patients with six repeat sequences [29]. UGT1A1*60 (in linkage disequilib-
rium with TATA box variants) and UGT1A1*93 are the other variants located in the promoter
region. Both of them are found homozygous in around 10% of Caucasians [30]. UGT1A1*6
and UGT1A1*27 are located in coding region exon 1. UGT1A1*6 is the most frequent variant
in Asian populations (not found in Caucasians) and associated with ~30% decreased enzyme
activity in homozygous patients. UGT1A1*27 is almost completely eradicated enzyme activ-
ity. Nearly 3% of Asian people, are homozygous for both *6 and *27 variant [28, 31].

2.2.2. Dihydropyrimidine dehydrogenase gene (DPYD)

The pyrimidine antimetabolites 5-fluorouracil and its oral prodrug capecitabine are widely
used chemotherapeutic agents in the management of variety of tumor types, including colorec-
tal, breast, and head and neck cancers. They activate metabolically and inhibit thymidylate
synthase enzyme, which takes role in cellular replication. However, 5-FU leads significant
toxicities, such as myelosuppression, mucositis, hand-foot syndrome, and diarrhea. On the
other hand, accumulation of knowledge on 5-FU mechanism has developed new strategies
that increase the treatment efficacy and response [32, 33]. 5-FU is metabolized by dihydropy-
rimidine dehydrogenase (DPD) enzyme, and it converts the fluoropyrimidine to its inactive
metabolite dihydrofluorouracil (Figure 3). DPD enzyme, which is encoded by DPYD gene, is
a rate-limiting enzyme of 5-FU catabolism and is also used for evaluating the variability of
5-FU metabolism among patients [34]. SNPs in the DPYD gene are responsible for insufficient
production of DPD enzyme; therefore, low levels of enzyme increase the half-life of the drug,
Genotype Ref SNP HGVS Drug Enzymatic activity
Region Clinical phenotype
UGT1A1*1 Common allele-Wild type (TA) 6TA Irinotecan Normal enzyme activity

UGT1A1*28 rs 8175347 (TA) 7 TA Irinotecan Reduced enzyme activity

TATA box *1/*28 Irinotecan dosing based on clinical
findings
*28/*28 Dose reduction recommended.

UGT1A1*36 rs 8175347 (TA) 5 TA Irinotecan Normal enzyme activity

TATA box Irinotecan dosing recommendations are less
clear.

UGT1A1*37 rs 8175347 (TA) 8 TA Irinotecan Normal enzyme activity

TATA box Irinotecan dosing recommendations are less
clear.

UGT1A1*60 rs 4124874 c.-3279T>G Irinotecan Normal enzyme activity

Promoter region Standard irinotecan dosing

UGT1A1*93 rs10929302 c.-3156G>A Irinotecan Normal enzyme activity

Promoter region Standard irinotecan dosing

DPYD*9A rs1801265 c.85T>C, p.Cys29Arg 5-fluorouracil Capecitabine Normal enzyme activity

Exon 2 Standard irinotecan dosing

DPYD*2A rs3918290 IVS14 + 1G>A 5-fluorouracil Capecitabine Reduced enzyme activity

Intron 14 and exon 14 Increased toxicity risk

DPYD*13 rs55886062 c.1679T>G, p.Ile560Ser 5-fluorouracil Capecitabine Reduced enzyme activity

Exon 13 Increased toxicity risk

TYMS 2R/2R, rs34743033 28bp VNTR (2R; 3R) 5-fluorouracil Capecitabine Enzyme activity based on the repeats
TYMS 2R/3RG, TYMS Promoter enhancer region 2R/2R: Decreased TYMS expression, increased
3RG/3RG, 5-FU responsiveness, increased risk of toxicity
2R/3RG, 3RG/3RG: Increased TYMS
expression, decreased 5-fluorouracil,
capecitabine responsiveness, poor prognosis.
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Genotype Ref SNP HGVS Drug Enzymatic activity

Region Clinical phenotype
TYMS 3RG/3RC, rs2853542 G>C SNP in 2nd repeat of 3R 5-fluorouracil Capecitabine Enzyme activity based on the repeats
TYMS 2R/3RC, allele (3RC) 3RG/3RC: Increased TYMS expression,
TYMS 3RC/3RC Promoter enhancer region decreased 5-FU responsiveness, poor prognosis
2R/3RC or 3RC/3RC: Decreased TYMS
expression, increased 5-FU responsiveness,
Genetic Polymorphisms

increased risk of toxicity

MTHFR, 677C>T rs1801133 c.677C>T, p.Ala222Val Methotrexate Reduced enzyme activity

Exon 4 Homozygosity: Risk for toxicity from drugs
and requirement for dosing adjustments/
discontinuation
Heterozygosity: Associated with intermediate
enzyme activity
Lower dose requirements for methotrexate

MTHFR, 1298A>C rs1801131 c.1298A>C; p.Glu429Ala Methotrexate Reduced enzyme activity

Exon 7 Homozygosity/Heterozygosity: Associated
with intermediate enzyme activity. Lower dose
requirements for methotrexate.

TPMT*1 Common allele-Wild type Common allele-Wild type 6-mercaptopurine; Normal enzyme activity
Azathioprine; Thioguanine

TPMT*2 rs1800462 c.238G>C, p.Ala80Pro 6-mercaptopurine; Poor activity

Exon 5 Azathioprine; Thioguanine Homozygosity: very low/absent enzyme
activity
Heterozygosity: intermediate activity
Low/Absent Activity: Increased risk for
developing life-threatening side effects at a
standard dose of a thiopurine drug.
Intermediate (Reduced) Activity: Increased
risk for toxicity at a standard dose of a
thiopurine drug.
Genotype Ref SNP HGVS Drug Enzymatic activity
Region Clinical phenotype
TPMT*3A rs1800460 c.[460G>A;719A>G], 6-mercaptopurine; Poor activity
rs1142345 p.[Ala154Thr;Tyr240Cys] Azathioprine; Thioguanine Homozygosity—Low/Absent Activity: Increased
Exon 7, 10 risk for developing life-threatening side effects
from a standard dose of a thiopurine drug.
Heterozygosity- Intermediate (Reduced)
Activity: Increased risk for toxicity from a
standard dose of a thiopurine drug.

TPMT*3B rs1800460 c.460G>A, p.Ala154Thr 6-mercaptopurine, Poor activity

Exon 7 Azathioprine, Thioguanine Homozygosity—Low/Absent Activity:
Increased risk for developing life-threatening
side effects at a standard dose of a thiopurine
drug.
Heterozygosity—Intermediate (Reduced)
Activity: Increased risk for toxicity at a standard
dose of a thiopurine drug.

TPMT*3C rs1142345 c.719A>G, p.Tyr240Cys 6-mercaptopurine, Poor activity

Exon 10 Azathioprine, Thioguanine Homozygosity—Low/Absent Activity: Increased
risk for developing life-threatening side effects
from a standard dose of a thiopurine drug.
Heterozygosity—Intermediate (Reduced)
Activity:
Increased risk for toxicity from a standard dose
of a thiopurine drug.

CYP2D6*1 Common allele-Wild type Common allele-Wild type Tamoxifen Normal activity
Extensive metabolizer

CYP2D6*2 rs16947 c.584G>c, p.Arg296Cys Tamoxifen Normal enzyme activity

Exon 2

CYP2D6*4 rs3892097 1846G>A Tamoxifen Inactive enzyme

Exon 3—Junction of intron 3
and exon 4
(Not applicable variant occurs
in a noncoding region)
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Genotype Ref SNP HGVS Drug Enzymatic activity

Region Clinical phenotype
CYP2D6*5 Whole gene deletion Tamoxifen Inactive enzyme
On allele

CYP2D6*6 rs5030655 c.1707delT, p.Trp152Gly Tamoxifen Inactive enzyme

Exon 3
Genetic Polymorphisms

CYP2D6*9 AGA deletion at 2613-2615, Tamoxifen Reduced enzyme activity

p.Lys281 del (Partially functioning)

CYP2D6*10 rs1065852 c.188C>T, p.Pro34Ser Tamoxifen Reduced enzyme activity

Exon 1

CYP2D6*17 rs28371706 c.320C>T, p.Thr107Ile; Tamoxifen Reduced enzyme activity

rs16947 c.886T>C, p.Cys296Arg
Intron 1

CYP2D6*41 rs28371725 c.2988G>A Tamoxifen Reduced enzyme activity

Intron 6
(Not applicable variant occurs
in a noncoding region)

Note: Guidelines for the description and nomenclature of gene variations are available from the Human Genome Variation Society (HGVS): http://www.hgvs.org/content/
guidelines.

Table 3. Biological impact of the important pharmacogenetic biomarker variants [28, 42–46].

 Polymorphisms in Pharmacogenetics of Personalized Cancer Therapy 15
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Figure 3. Metabolism of 5-fluorouracil (5-FU). The initial and rate-limiting enzyme of 5-FU is dihydropyrimidine
dehydrogenase (DPD), catalysing the conversion of 5-FU into 5,6-dihydrofluorouracil (DHFU). DHFU is degraded
into fluoro-beta-ureidopropionic acid (FUPA) and fluoro-beta-alanine (FBAL) through degradation cascade. 5-Fluoro-
2′-deoxyuridine-5′-monophosphate (5-FdUMP), is the cytotoxic product resulting from a multi-step activation of
5-FU, inhibits the enzyme thymidylate synthase (TS) enzyme. This inhibition leads to accumulation of deoxy-uridine-
monophospate (dUMP) and depletion of deoxy-thymidine-monophosphate (dTMP), which leads to inhibition of DNA
synthesis. 5-FU can also inhibit RNA synthesis in a pathway that involves 5-fluorouridine monophosphate (5-FUMP)
and subsequent conversion to 5-fluorouridine triphosphate (5-FUTP) via 5-fluorouridine diphosphate (5-FUDP).

thus, resulting in excess drug accumulation and toxicity due to the inefficient catabolism of
drug. Genetic testing of polymorphisms is being used for the classification of patients who
would be at high risk for severe or fatal toxicity when receiving fluoropyrimidine-based che-
motherapy (Table 3) [35]. Complete deficiency of DPD has been seen in approximately 5% of
the overall population and also 3–5% of the population has a partial DPD deficiency due to
sequence variations in DPYD gene [36].

The IVS14+ 1G>A change with the combination of a mutation in intron 14 and a deletion
at 5′-splice consensus sequence of exon 14, the most known and frequent variant (known as
DPYD*2A), is resulting the formation of a truncated enzyme product lacking activity. The
estimated incidence of homozygous genotype of this allelic variant is 0.1% and heterozy-
gous genotype is 0.5–2.0% in Caucasians [37, 38]. Other variants, which are associated with
increased toxicity risk, include 496A>G in exon 6, T1679G (DPYD*13) in exon 13 and 2846A>T
in exon 22 [39–41]. Genetic mutations in DPYD can be analyzed by highly sensitive methods
even for heterozygous variants such as pyrosequencing. But determination of the 5-FU and
dihydrofluorouracil concentration ratio in plasma by high-pressure liquid chromatography
(HPLC) may be more reliable predictor test for toxicity [35]. Besides DPYD, there are some
other pharmacogenetic biomarkers that are being used for the determination of the efficacy
and toxicity of fluoropyrimidine-related therapies, such as thymidylate synthase gene (TYMS).

2.2.3. Thymidylate synthase gene (TYMS)

The main intracellular target of fluoropyrimidine-related therapies is to inhibit thymidylate

synthase (TS) enzyme (encoded by the TYMS gene) that catalyzes the transformation of dUMP,
16 Genetic Polymorphisms

which is essential for DNA replication (Figure 3) [47]. Fluorodeoxyuridylate (5-FdUMP), an

activated metabolite from 5-FU, forms stable complexes with TS enzyme and folate to stop
DNA synthesis over blocking the conversion of dUMP to dTMP. Therefore, low expression
levels of TS enzyme in colorectal patients receiving 5-FU-based treatment were associated
with desired drug response as well as to longer survival [47]. Polymorphisms, which lead
TYMS variations (2R/2R, 2R/3R, or 3R/3R) by forming double-tandem repeat (2R) or a triple-
tandem repeat (3R) on the TYMS promoter enhancer region (generally abbreviated as TSER),
influence the translation of TYMS mRNA and toxicity (Table 3). It has been showed that
homozygous 3R/3R cells overexpressed TYMS mRNA compared with homozygous 2R/2R
cells [48]. Apart from these repeats, a G>C SNP has been showed on the 12th nucleotide of the
second repeat at 3R allele, and it is causing a tri-allelic locus as 2R, 3RG, and 3RC. The 3RC
allele has similar transcriptional activity of the 2R allele [49]. Another genetic polymorphism
of the TYMS gene has been identified as a 6 basepair deletion at position 1494 in the 3′-untrans-
lated region (3′-UTR). It was demonstrated that there is a strong association between the 6-bp
deletion and low TYMS mRNA expression in colorectal tumor tissue [50, 51].

2.2.4. Methylene tetrahydrofolate reductase (MTHFR)

Methylene tetrahydrofolate reductase (MTHFR) a regulatory enzyme is involved in folate

metabolism that redirects folate metabolites from pyrimidine synthesis towards methionine
synthesis (Figure 3). Most of the MTHFR gene variants of enzyme contain polymorphisms
that lead loss-of-function in the enzyme (Table 3). The level of loss-of-function depends on
the type and number of polymorphisms in coding gene. Therefore, MTHFR polymorphisms
affect drug metabolism, such as methotrexate, may be more likely to experience toxicity.
c.677C>T (p.A222V) and c.1298A>C (p.G429A) are the most common polymorphisms of
MTHFR gene that forms abnormal forms of enzyme. Both polymorphisms are associated
with reduced MTHFR enzyme activity. Adjustment drug dosages and prediction of the tox-
icity risk, MTHFR polymorphism test may be performed for a patient who is treated with
­methotrexate [23].

2.2.5. Thiopurine-S-methyltransferase (TPMT)

Thiopurine-S-methyltransferase (TPMT) enzyme, encoding by TPMT gene, catalyzes the

S-methylation of thiopurine drugs (such as 6-mercaptopurine, azathioprine, and thiogua-
nine) for drug inactivation. The purine antimetabolites are converted to active thioguanine
nucleotides that can be incorporated into DNA or RNA to block the DNA replication. During
this activation process, purine antimetabolites cause cellular toxicity for both malign and
benign cells. TPMT catalyzes inactivation of the formed thioguanine nucleotides [52]. It is
known that some patients have low enzyme activity due to germline genetic variations. There
are several TPMT polymorphisms that caused more than 20 variant alleles of TPMT gene
(from TPMT*2 to TPMT*20) and have different effects on TPMT function (Table 3). Among
these variant alleles TPMT*2, TPMT*3A, TPMT*3B, and TPMT*3C have been determined as
responsible for enzyme deficiency. Among these alleles TPMT*2, TPMT*3A and TPMT*3C are
deficient alleles that present poor enzyme activity [45]. Accumulation of cytotoxic t­ hiopurine
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nucleotides leading to severe toxicity has been shown in people with deficient TPMT alleles,
after receiving a standard dose thiopurine-based treatment. Patients with low TPMT activ-
ity expose more activated thioguanine nucleotides and more treatment toxicity as well as
efficacy. Patient’s genotype predicts thiopurine nucleotide levels and treatment outcomes in
thiopurine drug metabolism that shows tri-modal distribution with 89–94% of patients hav-
ing high enzyme activity, 6–11% of them having intermediate activity (as heterozygous), and
0.3% of them having low or no activity (as homozygous) [53].

2.2.6. Glucose-6-phosphate dehydrogenase (G6PD)

Glucose-6-phosphate dehydrogenase (G6PD) is a metabolic enzyme involved in pentose

phosphate pathway that is important in red blood cell metabolism. The G6PD gene is highly
polymorphic with more than 300 variants. G6PD gene deficiency is a common disease-caus-
ing enzymopathy and is associated with low levels of G6PD enzyme. The most remarkable
symptom of G6PD gene deficiency is hemolytic anemia caused by the ingestion of drugs and
food substances that result in oxidative stress. The excess peroxide due to deficiency has a risk
for both hemolytic and methemoglobinemia. Rasburicase is a recombinant urate oxidase that
has been used for the initial management of plasma uric acid in pediatric and adult patients
with leukemia, lymphoma and solid tumors who are receiving chemotherapeutic agents and
are expected for tumor lysis syndrome. Rasburicase is contraindicated in patients with G6PD
gene deficiency; therefore, FDA recommends screening for G6PD deficiency before beginning
rasburicase treatment. Dabrafenib is a kinase inhibitor that blocks the growth and spread of
cancer cells in the body. Dabrafenib consists of a sulfonamide moiety and contains hemo-
lytic anemia risk in patients with G6PD deficiency. Thus, FDA recommends monitoring for
patients with G6PD deficiency started with dabrafenib [54–56].

2.2.7. Cytochrome P450 2D6 (CYP2D6)

Cytochrome P450 2D6 (CYP2D6) deficiency is the first discovered pharmacogenetic deficiency
with the characterization of decreased enzyme expression-related polymorphisms. Decreased
mRNA levels of CY2D6 were determined as the reason of reduced metabolism and adverse
reactions to an anti-hypertensive drug, debrisoquine. These findings based on molecular tech-
nologies display the importance of genotyping on the pharmacological phenotyping involved
in drug metabolism [35]. CYP2D6 is a highly polymorphic enzyme (Table 3). According to
increased or decreased enzyme activity due to SNPs, duplications or deletions, genetic vari-
ants of CYP2D6 were classified into four metabolic phenotypes: ultra-rapid metabolizer,
extensive metabolizer, intermediate metabolizer and poor metabolizer (Table 4). This classifi-
cation system has been used to control treatment recommendations for several drugs, includ-
ing tamoxifen [57].
Tamoxifen is a selective estrogen receptor modulator, which is used in the treatment of estrogen
receptor-positive breast cancer. Tamoxifen is a weak estrogen antagonist by itself, but it is con-
verted into its main active metabolite, antiestrogenic endoxifen, by CYP2D6. CYP2D6 genotype is
predictive of endoxifen exposure that is critical in determining treatment outcome [59]. Increased
endoxifen exposure is associated with increased treatment efficacy and toxicity [60, 61].
18 Genetic Polymorphisms

Phenotype Genotype
Poor metabolizer Two inactive alleles

Intermediate metabolizer Two active alleles

Extensive metabolizer One active and one inactive alleles;

Two decreased activity alleles;
One decreased activity and one inactive allele

Ultra-rapid metabolizer More than two copies of active allele

Notes: Active alleles: *1, *2, *33, *35; decreased activity alleles: *9, *10, *17, *29, *36, *41; inactive alleles: *3, *4, *5, *6, *7,
*8, *11-*16, *19-*21, *38, *40, *42.

Table 4. CYP2D6 phenotypes according to activity status of CYP2D6 alleles (adapted from Swen JJ., et al. 2011) [58].

2.3. Biomarker discovery studies for pharmacogenetic applications

Pharmacogenetics and its backbone studies in terms of polymorphisms present new devel-
opments and trends in the field of tailored medications and advancements in the modifi-
cation of therapeutic choices utilizing genotypic information from polymorphism analysis.
Pharmacogenetic biomarker studies have multiple processes from discovery to clinical imple-
mentation (Figure 4). The ultimate aim of the biomarker studies is to find a clinically accessible
decision-maker biomarker to improve patient outcomes. However, many of the valid associa-
tions cannot be achieved for clinical implementation due to the lack of sufficient robustness
or clinical importance for the question. Biomarker discovery studies should be performed
as the screening of genotype-phenotype relations in large cohorts with statistical and bioin-
formatics tools. The most significant markers obtained from discovery studies are replicated
for analytical validation in different cohorts with the evaluation of assay reproducibility and
robustness. After successful analytical validity, the biomarker and assay must be evaluated
to confirm its performance in diagnosing the clinical phenotype or predicting outcome of

Figure 4. Schematic presentation of pharmacogenetic biomarker studies from development to clinical implementation.

 Polymorphisms in Pharmacogenetics of Personalized Cancer Therapy 19
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interest. A clinically valid biomarker assay can undergo translation through prospective con-
firmation of clinical utility to improve patient outcomes. And finally, analytically and clini-
cally validated biomarker assay is ready for implementation phase that includes regulatory
approval and incorporation in clinical practice guidelines, commercialization and coverage
by health insurance [52, 62].

2.4. Future directions and conclusion

After “Human Genome Project,” science world began to use individual genotypic informa-
tion to predict the risk of diseases, to prognose the disease, to guide therapeutic decision-
making, and to develop targeted medications. Especially, the better prediction ability for
drug response and life-threatening toxic reactions is highly crucial for clinicians due to the
narrow therapeutic index of many cancer chemotherapeutic agents. The aim of personal-
ized medicine is to prescribe a convenient chemotherapeutics to the right patients with right
dose to achieve maximal therapeutic benefit with minimal toxicity. Deep observation of the
human genome is still ongoing, and it will help to discover new targets and select the most
efficacious drug for each patient’s tumor, which is named as genomic prescribing system,
the next ­evolution of systemic cancer management. Genetic polymorphisms, as significant
biomarkers, have provided significant and crucial information in the management of toxic-
ity and dosing of cancer treatments. So far the achievements have been limited due to the
multifactorial challenges on the polymorphism-based personalized cancer management.
Especially reliable clinical data for the effects of genetic alterations on the disease patho-
genesis, drug metabolism and response are not always available; and performing the large-
scale prospective clinical studies, to understand the associations of polymorphisms and the
application of chemotherapeutics, is often laborious. However, these prospective studies are
required for establishing possible relations for evaluating the utility and cost-effectiveness of
polymorphism-based pharmacogenetic tests and personalized medicine. Ethical, social, and
regulatory issues relevant with pharmacogenetic-based personalized medicine are the other
complex and challenging factors to establish the relations between genetic polymorphisms
and personalized drug responses.
The main application of genetic polymorphism knowledge is improving the futuristic health
care through gene therapy, discovery of new drugs and drug targets and upgrade the discov-
ery processes with advanced technologies. The use of single candidate genes can be useful as
a part of initial treatment, but in near future, it will never be enough to provide fully tailored
treatment decisions on the cancer management. Other omics technologies will complement
the genotype-phenotype association-based pharmacological-pathway approach, such as tran-
scriptomics, epigenomics, and miRNAomics.

2.4.1. Polymorphism and pharmacogenetics facts

• Humans share about 99.9% sequence identity. The other 0.1% are mostly SNPs.

• SNPs are the most common polymorphism type and occur about every 1000 bases.
20 Genetic Polymorphisms

• SNPs can be silent—99% of them are not in coding regions and not in genes and thus cause
harmless, harmful, latent changes.

• Genetic polymorphisms are one of the most important factors that may contribute to ethnic
sensitivity of a drug.

• FDA recommends for following guidelines relating to “ethnically sensitive situations” for
some drugs and suggests solutions that may control such ethnic sensitivity in the context
of therapeutic applications and study designs.

• The Hardy-Weinberg principle can be used to calculate allele frequencies.

• Hardy-Weinberg principle-based reporting may be optimal in genetic polymorphism-

based studies.

• Personalized medicine separates patients into different groups with an individual’s mo-
lecular profile and covers the determination of the safest and most effective chemothera-
peutic agents.

• The aim of pharmacogenetics is to make ‘personalized medicine’ as applicable to various

patient groups.

• Although a number of good polymorphism genotyping technologies are currently in use

to meet the needs of clinics and researches, not only one genotyping method is ideal for
all applications. Studies on ideal polymorphism genotyping technologies are still ongoing.

• FDA has been working to improve pharmacogenetics technologies in the development,

regulation and use of medications and revise drug labels in terms of pharmacogenetic bio-
markers in oncology area.

• Polymorphism in the DPYD gene is responsible for insufficient production of DPD en-
zyme; therefore, low levels of enzyme increase drug accumulation and toxicity due to the
inefficient catabolism of drug.

• Allelic differences of UGT1A1 affect the glucuronidation of SN-38, active metabolite of iri-
notecan both in vitro and in vivo.

• TPMT polymorphisms have different effects on function of TPMT that inactivates thio-
guanine nucleotides. Patients with low TPMT activity expose more activated thioguanine
nucleotides and more treatment toxicity as well as efficacy.

• FDA recommends screening for G6PD deficiency before the beginning of rasburicase
treatment.

• CYP2D6 deficiency is the first discovered pharmacogenetic deficiency. Genetic variants of

CYP2D6 are classified into ultra-rapid metabolizer, extensive metabolizer, intermediate
metabolizer, and poor metabolizer.

• Biomarker development studies involve multiple steps, as biomarker assay development,

analytical validation, clinical validation, and clinical implementation.
 Polymorphisms in Pharmacogenetics of Personalized Cancer Therapy 21
http://dx.doi.org/10.5772/intechopen.69207

Author details

Gizem Calibasi Kocal1 and Yasemin Baskin1,2,3*

*Address all correspondence to: [email protected]

1 Department of Basic Oncology, Institute of Oncology, Dokuz Eylul University, Izmir, Turkey

2 Personalized Medicine and Pharmacogenomics/Genomics Research Centre-BIFAGEM,

Dokuz Eylul University, Izmir, Turkey

3 Faculty of Medicine, Department of Medical Informatics and Biostatistics, Dokuz Eylul

University, Izmir, Turkey

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