C 1

Chapter 1
Pharmacogenetic screening in cancer

treatment and cancer risk
Chapter 1.1
Genetic polymorphisms of drug
metabolising enzymes and drug transporters
in the chemotherapeutic treatment of cancer
Tessa M. Bosch
Irma Meijerman
Jos H. Beijnen
Jan H.M. Schellens
In press: Clinical Pharmacokinetics
3
Chapter 1.1
Abstract
There is a wide variability in the response of individuals to standard doses of drug therapy.
This is an important problem in clinical practise, were it can lead to therapeutic failures or
adverse drug events. Polymorphisms in genes coding for metabolising enzymes and drug
transporters can affect drug efficacy and toxicity. Pharmacogenetics aims to identify
individuals predisposed to high risk of toxicity and low response from standard doses of anticancer drugs. This review will focuss on the clinical significance of polymorphisms in drug
metabolising enzymes (CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, DPD,
UGT1A1, GST, SULT1A1, NAT, TPMT) and drug transporters (P-gp, MRP2, BCRP) in
influencing efficacy and toxicity of chemotherapy.
The most important example to demonstrate the influence of pharmacogenetics on anticancer therapy is thiopurine methyltransferase (TPMT). A decreased activity of TPMT,
caused by genetic polymorphisms in the TPMT gene, causes severe toxicity in the treatment
with 6-mercaptopurine (6-MP). Dosage reduction is necessary for patients with heterozygous
or homozygous mutation in this gene.
Other polymorphisms showing the influence of pharmacogenetics in chemotherapeutic
treatment in cancer, are discussed, such as UGT1A1*28. This polymorphism is associated
with an increase in toxicity in irinotecan treatment. Also, polymorphisms in the DPYD gene
show a relation with 5-fluorouracil-related toxicity. However, in most cases no clear
association has been found for polymorphisms in drug metabolising enzymes and drug
transporters, and pharmacokinetics or pharmacodynamics of anti-cancer drugs. The studies
discussed evaluate different regimens and tumour types and show that polymorphisms can
have different, sometimes even contradictory, pharmacokinetic and pharmacodynamic
effects in different tumours in response to different drugs.
The clinical application of pharmacogenetics in cancer treatment will therefore require
more detailed information of the different polymorphisms in drug metabolising enzymes and
drug transporters. Larger studies, in different ethnic populations, and extended with
haplotype and linkage disequilibrium analysis, will be necessary for each anti-cancer drug
separately.
16
Pharmacogenetic screening in cancer treatment and cancer risk
Introduction
Patients who are treated with standard doses of chemotherapy exhibit a large inter- and intraindividual variability in the development of severe toxicities. In addition, there is a wide
variation between patients with the same tumour type and stage in the likelihood of
response after standard chemotherapy.
The factors responsible for the individual pharmacokinetic and pharmacodynamic
variability are manifold and include drug-drug interactions, ethnicity, patients age, renal and
liver function, concomitant diseases, nutritional status, smoking and alcohol consumption.
In many cases, however, genetic factors have an even greater influence on drug efficacy and
toxicity.[1] Such variation, for instance seen in genes coding for drug metabolising enzymes
and drug transporters, can influence pharmacokinetic and pharmacodynamic parameters of
anti-cancer drugs.
Genetic variation in the human genome is a common phenomenon and approximately 1 out
of 1000 bp differs between any two individuals. Most of these variations are single nucleotide
polymorphisms (SNPs), a single nucleotide difference.[2] The number of polymorphisms
identified in genes encoding drug metabolising enzymes and drug transporters is rapidly
increasing, probably leading to a better explanation for the observed variation in efficacy
and toxicity of anti-cancer drugs in patients.
Phase I drug-metabolising enzymes, especially Cytochrome P450 (CYP), can activate or
inactivate the administered drug. Phase II metabolising enzymes, such as uridine
diphosphate glucuronosyl transferases (UGTs) and Glutathione S-Transferases (GSTs),
usually inactivate the active form of the drug or its metabolite by conjugation.
Polymorphisms in these enzymes can therefore result in a different pharmacokinetic and
pharmacodynamic profile of therapeutic agents. Polymorphisms in drug transporters, such
as P-glycoprotein, and other receptors can influence the uptake and excretion of a drug,
thereby also causing inter-individual variability in pharmacokinetics of that drug. The
pharmacodynamic profile can also vary between patients because the sensitivity of the
tumour for the chemotherapeutic agents can change if P-glycoprotein excretes the drug out
of the tumour-cell.
Pharmacogenetics is the study of the inherited basis of interindividual differences in drug
response. One approach is to search for genetic variants that are associated with severe
adverse effects, which, in turn, can be used to screen for individuals who should not receive
the drug in question or should receive an adjusted dose of the drug. Another approach is to
identify markers that predict drug efficacy.[3-5] Pharmacogenetic screening before the start of
cancer chemotherapy may thus enable the identification of patients at increased risk of
toxicity or low likelihood of response. The promise of pharmacogenetics is that it could lead
to tailored drug therapy, also called individualised medicine. This review will focus on the
clinically significant role of genetic polymorphisms of selected drug-metabolising enzymes
and adenosine triphosphate-binding cassette (ABC)-drug transporters and their possible
influence on toxicity and/or response of chemotherapy.
17
Chapter 1.1
Phase I enzymes
Cytochrome P450 (CYP)
Among the human CYP enzymes involved in metabolism of anti-cancer agents, genetic
polymorphisms have been reported in CYP2C8, 2C9, 2C19, 2D6, and 3A4 isoforms. The
effects of polymorphisms in these enzymes on the metabolism of anti-cancer drugs are
described below.
CYP2C8
Several mutation have been described in the CYP2C8 gene (see Table 1). CYP2C8*3 was
shown to be defective in the metabolism of paclitaxel, using cDNA expressed in Escherichia
Coli. The formation of 6--hydroxy paclitaxel in cells with the CYP2C8*3 polymorphism was
15% of that of wild-type cell lines. The intrinsic clearance of paclitaxel could not be
determined because of the low activity of CYP2C8*3.[6] Soyama et al.[7] and Bahadur et al.[8]
also found a reduced in vitro clearance for paclitaxel for CYP2C8*3 in HepG2 cell lines and
human liver microsomes respectively.
The CYP2C8 activity associated with CYP2C8*4 also appeared lower than the wild-type, but
the difference was not significant.[8] For CYP2C8*2, an increased Michaleis-Menten constant
for paclitaxel 6-hydroxylation has been observed.[6] Several intronic SNPs have been
identified at the intron-exon boundaries,[7] but whether these influence transcription rate or
mRNA splicing, or are otherwise biologically interesting, remains to be determined.
It remains to be determined whether individuals with mutant alleles (heterozygous or
homozygous) show decreased metabolism of paclitaxel, ifosfamide and cyclophosphamide
in vivo.
CYP2C9
CYP2C9 is 92% homologous to CYP2C19, the expressed product of its neighbouring gene
CYP2C19, differing by only 43 out of 490 amino acids. However, the two enzymes have
different substrate specificity. CYP2C9 is involved in the metabolism of the anti-cancer drugs
cyclophosphamide, ifosfamide, etoposide and tamoxifen (see Table 1 and 2).
In vitro data have consistently demonstrated that the CYP2C9*2 and *3 alleles are associated
with significant reductions in the intrinsic clearance compared with CYP2C9*1 of a variety of
2C9 substrates; however, the degree of these reductions appear to be highly substratedependent.[9]
Wild type CYP2C9 is more efficient than the mutant allele CYP2C9*3 in cyclophosphamide 4hydroxylation and ifosfamide 4-hydroxylation in in vitro studies.[10] Watters et al.[11] found no
correlation between genotype of Cyp3a13, the mice homologue for human CYP2C9 and
cyclophosphamide response. Currently no data on the in vivo effect of CYP2C9
polymorphisms on pharmacokinetics or pharmacodynamics of cyclophosphamide,
ifosfamide and tamoxifen are available.
18

Table 1: Characteristics of phase I drug metabolising genes (with the most important
polymorphisms)
Gene
Location
(chromosome)
Protein
Exons
Amino
acids
CYP2C8
10q24
CYP2C8
CYP2C9
10q24
CYP2C9
CYP2C19
CYP2D6
CYP3A4
CYP3A5
DPYD
10q24
22q13
7q22
7q22
1p22
CYP2C19
CYP2D6
CYP3A4
CYP3A5
DPD
13
13
23
Polymorphisms
Location
(Exon)
Effect
490
*2 (A805T)
*3 (A1196G, G416A)
*4 (C792G)
5
8, 3
5
I269F
K399R, R139K
I264M
490
*2 (C430T)
*3 (A1075C)
3
7
A144C
I359L
*2 (G681A)
*3 (G636A)
*4 (A1G)
Intron 5
4
1
*5 (C1297T)
*6 (G395A)
*7 (IVS5+T2A)
*8 (T358C)
haem
3
Intron 5
3
Splicing defect
Stop codon
Initiation
codon
R433W
R132Q
Splice site
W120R
491
*3 (2549delA)
*4 (G1846A)
*5 (gen deleted)
*6 (1707delT)
5
Intron 3
3
Frame shift
Splicing defect
Deleted
Frame shift
503
*1B (A>G)
*2 (T15713C)
*3 (T23172C)
*12 (C21896T)
5-UTR
7
12
11
S222P
M445T
L373F
502
*3 (A6986G)
*5 (T12952C)
*6 (G14690A)
*7 (27131-32insT)
Intron 3
?
?
?
Splicing defect
Splicing defect
Splicing defect
Frame shift
1025
GIVS14+1A
C61T
G62A
T85C
A496G
G1003T
G1156T
G1601A
A1627G
T1679G
A2194A
A2846T
14
2
2
2
6
9
10
13
13
13
18
22
Skip exon 14
R21X
R21Q
C29R
M166V
V335L
E386X
S534N
I543V
I560S
V732I
D949V
490
19
20
DPD*
-
Methotrexate
Cyclophosphamide
DPD*
CYP2C9, CYP3A4/5
Etoposide
Capecitabine
5-Fluorouracil
CYP3A4/5*
Carboplatin
Topotecan
Cisplatin
Irinotecan
Oxaliplatin
TPMT, GST
UGT1A1, GSTP1
UGT1A1
GSTM1, GSTP1, GSTT1
GSTM1, GSTP1, GSTT1
GSTM1, GSTP1, GSTT1
UGT1A1, UGT2B7
GSTP1
GST
Phase II metabolising
enzymes
CYP2A6, CYP2B6,*
GSTA1
CYP2C8/9/19, CYP3A4/5
Ifosfamide
CYP2A6, CYP2B6,*
GST
CYP2C8/9/19, CYP3A4/5
* major contribution of metabolising enzyme to metabolism of this anti-cancer drug.
Alkylating agents
Anti-metabolites
Topo-isomerase inhibitors
Platinum consisting agents
CYP3A4/5*
Vincristine
Epirubicin
CYP3A4/5*
Docetaxel
Doxorubicin
CYP2C8*, CYP3A4/5*
Paclitaxel
Anti-mytotic cytostatics
Anti-tumour antibiotics
Phase I metabolising
enzymes
Anti-cancer drug
Table 2: Anti-cancer drugs and their drug metabolising enzymes and drug transporters
MRP2, BCRP
P-gp
P-gp, MRP2, BCRP
P-gp, MRP2, BCRP
MRP2
MRP2
MRP2
P-gp, MRP2, BCRP
P-gp, BCRP
P-gp
P-gp
P-gp
Drug transporters

CYP2C19
Individuals can be characterized as extensive metabolisers or poor metabolisers of drugs
metabolised by CYP2C19. Poor metabolisers represent 2-5% of Caucasian and 13-23% of
Asian populations. African populations have been studied less extensively but the poor
metaboliser trait has been reported to be approximately 4%. The most common defective
allele are mentioned in Table 1.
In a study with prostate cancer patients treated with thalidomide, the pharmacogenetics of
CYP2C19, responsible for 5-hydroxylation and 5-hydroxylation of thalidomide, has been
studied. In two patients with the homozygous CYP2C19*2 polymorphism (poor
metabolisers) no quantification of the 5-hydroxythalidomide and cis-5-hydroxythalidomide
could be detected.[12]
However, to date, in general it seems that the polymorphisms in this gene have no
measurable effect on the anti-cancer therapy in vivo.
CYP2D6
CYP2D6 activity is absent in 5-10% of European and north American Caucasian
populations.[13] The CYP2D6 gene is one of the best studied human Cytochrome P450s, and
variant alleles range from *1 (wild type) to *51, with 26 identified null alleles and 6 alleles
encoding enzymes with decreased activity (see http://www.imm.ki.se/CYPalleles/
cyp2d6.htm accessed Sept 30th, 2004). In addition, gene duplication is responsible for the
occurrence of an ultra rapid CYP2D6 metabolism. The most common alleles in the
Caucasian population are CYP2D6*3, *4, *5 and *6, and none of these four variants produce
functional CYP2D6 (see Table 1). With respect to anti-cancer drugs, CYP2D6 is only involved
in the conversion of tamoxifen to the 50 times more potent anti-estrogen 4OH-tamoxifen.[14]
Plasma endoxifen concentrations (a metabolite of tamoxifen) after 4 months of tamoxifen
therapy were statistically significantly lower in subjects with a CYP2D6 homozygous variant
genotype or a heterozygous genotype than in those with a homozygous wild-type genotype.
Besides, in subjects taking a potent CYP2D6 inhibitor (such as paroxetine) the plasma
endoxifen concentration was reduced substantially.[15] Although the role of CYP2D6 in
tamoxifen treatment is evident, most anti-cancer agents are not subjected to CYP2D6
metabolism at clinically relevant concentrations.[16]
CYP3A4
The allelic variants of CYP3A4 in humans have been investigated thoroughly (see
http://www.imm.ki.se/CYPalleles/cyp3a4.htm and Table 1). The CYP3A4*1B mutation was
found in different racial groups. The frequency of this allele in a white population varied
from 3.6 - 9.6%,[17-22] whereas it was 53-67% in a black population. The CYP3A4*1B allele was
not identified in Asian subjects. Goh et al.[23] did not find polymorphisms in the 5-flanking
region of the CYP3A4 gene in 32 patients treated with docetaxel. Besides, no association has
been found between the CYP3A4*1B allele genotype and pharmacokinetics of
substrates.[17,20,24,,25]
21
Chapter 1.1
A second variant allele, CYP3A4*2, has been found in a frequency of 2.7% in a white
(Finnish) population[19] and has been shown to be absent in black and Asian subjects. This
variant had a reduced intrinsic clearance for nifedipine compared to the wild-type, while the
testosterone 6-hydroxylase activity and pharmacokinetics were not significantly different
from the wild-type.[20]
Another variant allele, CYP3A4*3, has been identified in 1-4% in white subjects[19,26] and in
one single Chinese subject.[20] This allelic variant is located at a conserved region that
contains the cystein residue that serves as the fifth ligand of the heme iron in the P450 active
site. For CYP3A4*3 no correlation between genotype and metabolism of testosterone,
progesterone and chlorpyrifos has been observed.[26,27] Eiselt et al.[27] have identified a new
CYP3A4 allele variant, CYP3A4*12, with a frequency of 0.34% in Caucasians, that showed
altered enzyme activity for testosterone and midazolam.
Regulation of CYP3A4 expression is essentially pretranslational and its mRNA levels allow a
good estimate of its activity.[28] The polymorphisms in the CYP3A4 gene however cannot
explain the great variability in expression of CYP3A4.
The expression of CYP3A4 has been associated with clinical outcome of anti-cancer
treatment. Patients with low CYP3A4 mRNA levels in tumour tissue exhibited significantly
higher response rate to docetaxel treatment in breast cancer patients than those with high
CYP3A4 mRNA levels. However, no significant association has been observed between
CYP3A4 mRNA expression and response to cyclophosphamide and epirubicin.[29]
The CYP3A4*1B allele is associated with a reduced risk for treatment-related leukaemia after
treatment of the primary cancer with epipodophyllotoxin therapy.[30] Patients with the wild
type genotype have an increased production of potentially DNA-damaging reactive
intermediates compared with CYP3A4*1B. The mutant alleles may decrease production of
the epipodophyllotoxin catechol metabolite, which is the precursor of the potentially DNAdamaging quinone.
No association has been found between CYP3A genetic variants and an increased risk of
relapse in patients with acute lymphoblastic leukaemia (ALL).[31] However, patients with
CYP3A4*1B genotype had a statistical significant decreased risk of peripheral neuropathy in
a univariate analysis. After correction for multiple comparisons, the association approached,
but did not reach, statistical significance. In another study, therapy-related acute myeloid
leukaemia (AML) in children treated for AML has not been associated with CYP3A4*1B
genotype.[32]
All of the coding Single Nucleotide Polymorphisms (SNPs) allele frequencies of the CYP3A4
gene are relatively low in most of the populations studied and no homozygotes for these
mutations have been reported.[33] It was estimated that only 14% of Caucasians, 10% of
Japanese and 15% of Mexicans carry a CYP3A4 allele with at least one coding change. With
such low allele frequencies, it will be exceedingly difficult to link genotype to
22

pharmacokinetics and pharmacodynamics of CYP3A4 substrates. Maybe an explanation can
be given by polymorphisms in the Pregnane X Receptor (PXR) gene that regulates the
transcription of CYP3A4. Several studies have described polymorphisms in the PXR gene and
their influence on CYP3A4 expression. Hustert et al.[34] expressed three protein variants
(V140M, D163G and A370T) in LS174 cells and these proteins exhibited altered basal and/or
induced transactivation of CYP3A promoter reporter genes. Koyano et al.[35] also analysed
variant proteins (R98C, R148Q, R381W and I403V) and showed varying degrees of reduction
in transactivation, depending on the dose of PXR activators. Further research will be
necessary to identify the effects of PXR polymorphisms on the interindividual variability of
pharmacokinetics and pharmacodynamics of substrates for CYP3A4. Another possibility to
clarify the variability in pharmacokinetics and pharmacodynamics of CYP3A4 substrates is
linkage of CYP3A4 and CYP3A5 polymorphisms. Strong linkage disequilibrium (LD) has
been confirmed between CYP3A4 and CYP3A5, but so far no effects on pharmacokinetics
and pharmacodynamics have been described.[36]
Overall, no major pharmacokinetic consequences for the identified CYP3A4 mutations have
been observed for the metabolism of anti-cancer drugs. Therefore, the role of CYP3A4 on the
interindividual variability in anti-cancer therapy has to be investigated in the future with a
focuss on the LD and haplotype analysis and the function of transcriptional factors such as
the PXR.
CYP3A5
CYP3A5 represents at least 50% of the total hepatic CYP3A content in humans. The CYP3A5
protein is polymorphically expressed in adults with readily detecTable expression in about
10-20% in Caucasians, 33% in Japanese and 55% in African-Americans. The primary causal
mutation for the polymorphic expression (CYP3A5*3, see Table 1) confers low CYP3A5
protein expression as a result of improper mRNA splicing and reduced translation of the
functional protein. The CYP3A5*3 allele frequency varies from approximately 50% in AfricanAmericans to 90% in Caucasians.[37] Functionally, microsomes isolated from the livers of
homozygous mutants contain a very low amount of CYP3A5 protein and display an on
average reduced catalytic activity towards midazolam.
In a study of 25 patients, 9 patients were homozygous for the CYP3A5*3 genotype, and had
lower mean clearance of midazolam, but not docetaxel.[23] Also no significant difference in
docetaxel clearance was observed for the CYP3A5*3 genotype compared to wild-type in a
study of Puisset et al.[38] Kishi et al.[39] studied the effects of prednisone and genetic
polymorphisms in CYP3A5 on etoposide disposition in children with ALL. The CYP3A5*3
allele predicted a lower clearance of etoposide in black children. The CYP3A5*3 allele was
also associated with a statistical significant decreased risk of peripheral neuropathy in
patients with ALL in an univariate analysis.[31]
Several other CYP3A5 coding variants have been described, but they occur at relatively low
allelic frequencies and their functional significance has not been established.[33,40] Additional
intronic and exonic mutations (CYP3A5*5, *6, and *7) may alter splicing and result in a
23
Chapter 1.1
premature stop codon or exon deletion. Experiments with expression of these mutations in
E. Coli showed a lower testosterone clearance and a lower maximum rate (Vmax) for
nifedipine oxidation for mutant alleles compared with wild-type.[40]
In conclusion, to date no polymorphisms in the CYP3A5 gene have been described that
clarify the great variability in metabolism of anti-cancer drugs that are substrate for CYP3A5.
Transcriptional factors or linkage with other genes, such as CYP3A4, may possibly explain
the interindividual variability in patients.
Dihydropyrimidine dehydrogenase (DPD)

Dihydropyrimidine dehydrogenase (DPD) is the first and rate-limiting enzyme in the threestep metabolic pathway involved in the degradation of the pyrimidine bases uracil and
thymine. In addition, this catabolic pathway is the only route for the synthesis of -alanine in
mammals. Defects in the DPYD gene are a possible cause of DPD deficiency, also known as
hereditary thymine-uraciluria or familial pyrimidinaemia, which is characterized by
persistent urinary secretion of excessive amounts of uracil, thymine and 5hydroxymethyluracil, and accumulation of these compounds in blood and cerebrospinal
fluid.[41]
5-Fluorouracil (5-FU) is a major drug in the treatment of advanced colorectal cancer. Seventy
to 80% of the administered 5-FU is degraded by DPD in vivo to dihydrofluorouracil. Therefore
the activity of the enzyme DPD is one of the main factors determining exposure to 5-FU.
Patients with partial deficiency of this enzyme are at risk of developing severe 5-FUassociated toxicity as DPD is responsible for detoxification of pyrimidine-based antimetabolite analogues, such as 5-FU and capecitabine. The onset of toxicity occurs, on
average, twice as fast in patients with low DPD activity compared with patients with a normal
DPD activity.[42-45] A study of Di Paolo et al.[46] concluded that DPD activity in peripheral blood
mononuclear cells is unrelated to 5-FU/5-fluoro-dihydrouracil (5-FUH2) (the DPD dependent
metabolic product of 5-FU) pharmacokinetics. Only two of the five patients experiencing
severe toxicity had an abnormally low DPD activity, while in the remaining three subjects the
enzyme activity was normal. Also, it was shown that not all patients with low DPD activity
experience moderate or severe toxicity, as exemplified in a study in which 16 out of 84
patients who tolerated the treatment well had a low to normal enzyme activity.[46] Fleming et
al.[45] showed, however, a good correlation between 5-FU clearance and the DPD activity in
peripheral blood mononuclear cells and Etienne et al.[47] found a weak correlation. Thus,
overall, no clear correlation between DPD activity and 5-FU clearance could be detected.The
evaluation of DPD activity may therefore not be fully predictive of the risk of toxicity, as
pointed out before.
Another reason for the variation in 5-FU-related toxicity may be the detection of mutations in
the DPYD gene. Most patients with 5-FU-related toxicity have multiple mutations in the DPYD
gene (see Table 1). However, only a few patients with a low DPD phenotype have a
molecular basis for reduced activity. Although novel DPYD variants have been identified in
24

studies, the DPYD mutations now described do not entirely explain polymorphic DPD
activity and toxic response to 5-FU.
Analysis of the DPYD gene of a cancer patient that exhibited grade 4 toxicity 10 days after 5FU treatment revealed the presence of a splice site mutation GIVS14+1A, which lead to the
skipping of exon 14 directly upstream of the mutated splice donor site during DPD pre-mRNA
splicing.[48] These results are in line with other study results.[49-56] Among 25 patients with
severe 5-FU-related toxicity, 5 were heterozygous and 1 was homozygous for this mutation.
All of these patients had experienced grade 4 leukopenia and lethal outcome was seen in
the homozygous and two of the heterozygous patients.[53] A study of Maring et al.[56] indicated
that the inactivation of DPD by one heterozygous allelic mutation of GIVS14+1A in exon 14
can result in a strong reduction in 5-FU clearance, causing severe 5-FU induced toxicity. The
allele frequency of this mutation is 0.91% in a Dutch Caucasian population.[52] In patients with
low DPD activity, 42% were heterozygous and 3% was homozygous for this mutation.[54]
Almost similar results have been described by another study of van Kuilenburg et al.[57] A
lethal 5-FU toxicity was described in a patient with the GIVS14+1A mutation in combination
with a novel C61T nonsense mutation.[58] This nonsense mutation leads to a premature
termination of translation before the flavine adenosinedinucleotide (FAD) and
uracil/thymine binding sites and thus to a non-functional protein without any residual
activity.
In addition, other variants, such as T85C, A496G, G1601A, A1627G, T1679G and A2846T (see
Table 1), were also related to the reduced enzyme activity in patients.[50.59,60] A study of van
Kuilenburg et al.[50] showed that at least 57% of the patients with reduced DPD activity had a
molecular basis of their different phenotype.[50] In a study of Collie-Duguid et al.[59] only 17%
of the patients with a low DPD activity had a molecular basis for the reduced activity.
Other studies showed that the mutations T85C, A496G, G1601A, A1627G and A2194A had no
association with DPD activity.[60-62]
The first Japanese patient with decreased DPD activity, accompanied by severe 5-FU toxicity,
has been presented in a study of Kouwaki et al.[63] In a study of Yamaguchi et al.[64] only
adverse events of grade 1 or 2 were observed in Japanese patients treated with 5-FU,
suggesting that heterozygous mutations observed in these patients (such as T85C and
A1627G) had no association with toxicity of 5-FU. See Table 3 for all clinical effects of
polymorphisms in the DPYD gene.
These data emphasize the complex nature of the molecular mechanisms controlling
polymorphic DPD activity in vivo. The clinical utility for genetic polymorphism testing to
date is not optimal due to its low sensitivity and unknown specificity.[65] Also, the DPD
activity determination in peripheral blood mononuclear cells has no conclusive result in
identifying patients at risk of 5-FU toxicity. Overall, it can be remarked that the populations
examined are too small and probably, multiple mutations in the DPYD gene are responsible
for DPD deficiency and increased risk for 5-FU toxicity. The conflicting data show a need for
further research.
25
26
V335L
E386X
S534N
G1003T
G1156T
G1601A
I560S
Skip
exon 14
V732I
D949V
T1679G
GIVS14+1
A
G2194A
A2846T
*2
*6
I543V
M166V
A1627G
C29R
A496G
R21X
R21Q
C61T
G62A
T85C
Amino
acid
Nucleotide
*13
*5
*9A
Mutation
[60]
[50]
[60]
[63]
[63]
[59]
[50]
[59]
DPD activity and 5-FU toxicity

In combination with GIVS14+1A grade 4 toxicity after 5-FU combination treatment *
Leukopenia and severe mucositis during 5-FU treatment, DPD activity
Grade 4 toxicity after 5-FU treatment
No detecTable DPD enzyme
DPD activity and severe 5-FU toxicity
Lethal toxicity after 5-FU in homozygous mutant patient in Dutch population
6/25 patients with severe 5-FU toxicity had mutation and grade 4 leukopenia, of which 3 lethal
42% of patients with DPD activity was heterozygous, 3% homozygous
Gastrointestinal and haematological toxicity of 5-FU
clearance of 5-FU causing severe 5-FU toxicity
36% of patients with DPD activity was heterozygous, 4% homozygous
In combination with C61T lethal toxicity during 5-FU treatment*
In combination with A496G grade 4 toxicity after 5-FU combination treatment*
[50]
[50]

[48]
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[60]
[58]
[63]
Ref
In combination with GIVS14+1A lethal toxicity during 5-FU treatment*
Clinical effect
* Clinical effects referred to more than one genotype
Pharmacokinetic &
Pharmacodynamic
effects
Phenotype
Table 3: Effects of polymorphisms in the DPYD gene

Besides DPD, also thymidilate synthase (TS) and thymidine phosphorylase (TP) are involved
in the responsiveness to 5-FU. It has been shown that high gene expression of TS and TP in
pretreatment biopsies could identify tumours that are nonresponsive to 5-FU-based therapy.
In a study of Salonga et al.[66] the DPYD gene expression levels in tumours that were
unresponsive to 5-FU was much broader compared with that of responding tumours. All of
the tumours that respond to 5-FU therapy had expression values of all three of the genes, TS,
TP and DPD, below their respective nonresponse cutoff values, whereas, in each of the
nonresponding tumours, at least one of these genes had a high expression level.
Another enzyme in the catabolism of 5-FU is dihydropyrimidase (DHP). It has been
suggested that patients with a deficiency of this enzyme are at risk of developing toxicity
after 5-FU treatment. Van Kuilenburg et al.[67] demonstrated for the first time that in a patient
with severe 5-FU-related toxicity partial deficiency of DHP was responsible. Analysis of the
DHP gene showed that the patient was heterozygous for the missense mutation G883A in
exon 5 (G278D). The effects of polymorphisms in this gene for 5-FU related toxicity have to
be studied in more detail.
Phase II enzymes
Uridine diphosphate glucuronosyltransferase (UGT)
UGT1A1
UGT1A1 is capable of glucuronidating bilirubin. The clinically relevant polymorphisms
related to genetic abnormalities in the UGT1A1 enzyme are those associated with familial
(un)conjugated hyperbilirubinaemia syndromes such as Crigler-Najjar (type I and II) and
Gilberts syndrome. More than 50 genetic lesions in UGT1A1 have been reported, many of
which are found in patients with Gilberts syndrome.
UGT1A1 has an important role in the metabolism of irinotecan, etoposide, epirubicine and
tipifarnib (see Table 2). Irinotecan is a camptothecin derivative used in the treatment of
metastatic colon cancer. Irinotecan is a prodrug, since it is activated to SN-38 by carboxyl
esterase in order to exert its anti-tumour activity mediated by the inhibition of topoisomerase
I. SN-38 undergoes UGT1A1 catalysed glucuronide conjugation to form the inactive SN-38
Glucuronide (SN-38G).
In the promoter region of the UGT1A1 gene a micro satellite mutation has been found;
(TA)7TAA instead of (TA)6TAA (UGT1A1*28) resulting in reduced UGT1A1 expression levels
and activity. The frequency of the (TA)7 allele was 32-39% in Caucasians,[68-71] 40-43% in
Africans,[69,71] and 16-33% in Asians.[68,69,71] Alleles with five and eight TA repeats, i.e. (TA)5 and
(TA)8, have also been identified (respectively UGT1A1*33 and 34), although almost
exclusively in an African population with an allele frequency of 3.5 and 6.9%,
respectively.[68,69] The first case of a Caucasian subject affected by Gilberts syndrome and
heterozygous for the (TA)8 allele was described by Iolascon et al.[72]
27
Chapter 1.1
Homozygosity for UGT1A1*28 is usually associated with Gilberts syndrome, a mild chronic
unconjugated hyperbilirubinaemia, which often remains undiagnosed. Two patients with
metastatic colon cancer and Gilberts syndrome were treated with irinotecan-based
chemotherapy. Both patients presented grade 4 neutropenia and/or diarrhoea in every
treatment cycle.[73] A different metabolic ratio, SN-38/SN-38 Glucuronide (SN-38G), is a
possible explanation for interindividual variability of the pharmacokinetic profile of SN38
and its glucuronide.[74] This metabolic ratio in a patient with (TA)7/(TA)7 genotype was higher
than those in the patients with other genotypes ((TA)6/(TA)7 and (TA)6/(TA)6).[74] A significant
trend toward a decrease in SN-38 and bilirubin glucuronidation rates was found as the
number of TA repeats increased ((TA)6/(TA)6 > (TA)6/(TA)7 > (TA)7/(TA)7).[70,75-77] More severe
grades of diarrhoea and neutropenia were reported in patients heterozygous or homozygous
for the (TA)7 sequence.[76,78,79] Standard dosing regimens given to patients with Gilberts
syndrome with mild hyperbilirubinaemia display an increased area under the concentration
versus time curve (AUC) of SN-38/SN-38G, a factor that is linked to leukopenia (see Table
4).[78,80]
In contrast, in paediatric patients receiving irinotecan, no increased rate of grade 3-4
diarrhoea and neutropenia was observed in (TA)6/(TA)7 or (TA)7/(TA)7 patients. In part, this
may be due to the low dose protracted schedule utilised in this population (50 mg/m2/d * 5
q21d).[81] In Chinese nasopharyngeal carcinoma patients also no statistically significant
differences in the AUC values for irinotecan, SN-38 and SN-38G have been found in patients
with different UGT1A1*28 genotypes.[82] In a study of Marcuello et al.[79] no statistical
significance could be detected between UGT1A1 genotype and clinical response or overall
survival.
In black children with ALL who were treated with etoposide, the wild type UGT1A1*28
genotype ((TA)6) has been associated with a higher clearance of etoposide, also indicating
that the mutant genotype is responsible for a less potent glucuronidation.[39]
Other polymorphisms have been identified (see Table 5): In a study of Ando et al.[78] 3
patients heterozygous for UGT1A1*27 encountered severe toxicity of irinotecan, such as
leukopenia grade 4 and/or diarrhoea grade 3, or worse. For UGT1A1*6 the difference in
allelic distribution between patients with and without severe toxicity was not significant.
Thus the genotype of UGT1A1*6 is not related to the severity of the toxicity induced by
irinotecan.[78,80] However, a study of Sai et al.[83] identified several UGT1A1 polymorphisms
significantly associated with reduced AUC SN-38G/SN-38 ratios (*28 and *6) and with
increased total bilirubin level.
Other polymorphisms found, such as UGT1A1*7 and UGT1A1*29, did not affect the
pharmacokinetics or severity of the toxicity of irinotecan. The G-3156A variant seemed to
distinguish different phenotypes of total bilirubin within the TA tandem repeats genotypes.
28
29
(TA)6
(TA)7
C686A
*27
G211A
G-3156A
Nucleotide
*28
*6
Mutation
P229Q
G71R
Amino
acid
62.1
37.5
33
Caucasian
42.8 J
30.8 J
8.7 J
16.7 J
Other
Frequency (%)
SN-38/SN-38G 7/7 > 6/7 or 6/6

Glucuronidation rates 6/6>6/7>7/7
Enzyme activity: 6/6>6/7>7/7
Correlation between ANC and UGT1A1*28
(6/6 > 6/7 > 7/7) in patients treated with irinotecan
More severe grades of diarrhea and neutropenia in patients with 6/7and
7/7 with irinotecan
Patients with severe toxicity of irinotecan
Patients without severe toxicity of irinotecan
Severe diarrhea was observed in 70% 7/7 and 33% 6/7 and 17% 6/6
Value of AUCSN-38/AUCSN-38G above 75th percentile causing leukopenia
Reduced AUC SN-38G/SN-38 ratios in combination with *28 or *6
Grade 4 neutropenia after irinotecan treatment 6/6 0%, 6/7 12.5%, 7/7
50%
CL of etoposide in 6/7 or 7/7 genotype in week 54 of treatment in black
patients and AUC of the catechol metabolite in all patients
Severe toxicity irinotecan leukopenia grade 4 or diarrhea grade >3
Reduced AUC SN-38G/SN-38 ratios in combination with *28 or *6
Significant predictor of ANC after irinotecan treatment
Clinical effect
Abbreviations: J=Japanese, ANC=Absolute Neutrophil Count, AUC=area under the concentration time curve
Pharmacokinetic &
Pharmacodynamic
effects
Phenotype
Table 4: Clinical effects of SNPs in the UGT1A1 gene
[78]
[39]
[79]
[80]
[83]
[84]
[78]
[77]
[74]
[70]
[75]
[76]
[83]
[84]
Ref
Chapter 1.1
Table 5: Characteristics of phase II drug metabolising genes
(with the most important polymorphisms)
Gene
Location
(chromo
-some)
Protein
Exons
Amino
acids
UGT1A1
2q37
UGT1A1
532
GSTA1
6p12
GSTA1
222
GSTM1
GSTM3
1p13
1p13
GSTM1
GSTM3
7
9
218
225
GSTP1
11q13
GSTP1
210
GSTT1
22q11
16p1112
GSTT1
240
SULT1A1
295
NAT1
8p22
NAT1
290
NAT2
8p22
NAT2
290
TPMT
8p22
TPMT
245
SULT1A1
Polymorphisms
Location
(Exon)
Effect
G-3156A
T-3263G
(TA)6>(TA)7
(TA)6>(TA)5
(TA)6>(TA)8
*7 (C686A)
*6 (G211A)
*7 (T1456G)
*9 (C1099G)
*B (T-631G, T567G, C-69T, G-52A)
*0
*B (3 bp del)
*B (A313G)
*C (A313G, C341T)
Promoter
Promoter
Promoter
Promoter
Promoter
1
1
5
4
Promoter
P229Q
G71R
Y486G
R367G
-
Intron 6
5
5, 6
*0
*2 (G638A)
No gene
I105V
I105V,
V113A
No gene
H213R
*14 (G560A)
*17 (C190T)
*5 (T341C)
*6 (G590A)
*7 (G857A)
*10 (G499A)
*14 (G191A)
*17 (A434C)
*18 (A845C)
*19 (C190T)
*2 (G238C)
*3A (G460A, A719G)
4
4
2
2
2
2
2
2
2
2
4
6, 9
*3B (A719G)
*3C (G460A)
9
6
R187Q
R64W
I114T
R197Q
L268R
E167L
R64Q
Q145P
L282T
R64W
A80P
A154Y,
Y240C
Y240C
A154Y
The 3156 genotype and the AUC of SN-38 were significant predictors of absolute neutrophil
count nadir after irinotecan treatment.[84] The genetic variant T-3263G in the phenobarbital
responsive enhancer module of the UGT1A1 promoter region is also associated with
decreased transcriptional activity.[85] However, this study showed no association of this T3263G variant and severe toxicity of irinotecan.
Besides UGT1A1, another UGT isoform, UGT1A7, has been reported to glucuronidate SN-38 at
a more than 9-fold higher level compared to UGT1A1. This was demonstrated in vitro using
human UGT1 enzymes transiently expressed in COS-1 cells.[86] Recent pharmacogenetic
30

analysis, however, did not reveal a significant association between UGT1A7 genotypes and
toxicity caused by irinotecan.[87,88] The reason for this discrepancy is unclear, but may be
caused by expression of an abundant amount of UGT1A1 in the liver, compensating for low
amounts of UGT1A7. This suggests that UGT1A7 plays a minor role in SN-38 glucuronidation
in vivo.[80]
UGT1A10 has also been shown to catalyse the glucuronidation of SN-38.[89] However, the
contribution of UGT1A10 in the variability of irinotecan pharmacokinetics may be ruled out
because this isoform is also absent in human liver.[90]
In conclusion, the homozygous or heterozygous genotype for UGT1A1*28 may be a
significant risk factor for severe irinotecan toxicity. The example of irinotecan demonstrates
clearly how polymorphisms in an inactivating metabolic pathway may affect the adverse
events and therapeutic outcome in cancer chemotherapy.
Glutathione S-Transferase (GST)

GSTA1
GSTA1 has a high activity in glutathione conjugation of metabolites of cyclophosphamide.
Recently a polymorphism, has been described that alters hepatic expression of GSTA1, i.e.
GSTA1*B (see Table 5).[91] The survival in breast cancer patients treated with
cyclophosphamide-containing combination chemotherapy was reported to be associated
with the GSTA1*B polymorphism. Among patients who had no or one GSTA1*B allele, the
proportion surviving at 5 years was 66%, whereas for homozygous mutant GSTA1*B subjects
the proportion was higher, 86%.[92] See Table 6 for clinical effects of polymorphisms in the
GST genes.
GSTM1
No studies have been published so far that associate the effect of GSTM1 polymorphism with
the pharmacokinetics of anti-cancer drugs. Lack of GSTM1 activity, caused by an inherited
homozygous deletion of the entire GSTM1 gene (null genotype or *0), has been associated
with therapy outcome.The clinical response of treatment with anti-cancer drugs is influenced
by polymorphism in the GSTM1 gene, as was presented in several studies, however the
results have been inconsistent.
In a study from Stanulla et al.[93] the null genotype for GSTM1 conferred a 2-fold reduction in
the risk of relapse in childhood ALL patients after prednisone treatment. However, GSTM1
did not show any association with the response on prednisone treatment in children with
ALL treated according to protocols of the Berliner-Frankfurt-Munster study group.[94] A study
of Chen et al.[95] among childhood patients with ALL treated with teniposide, cytarabine and
high-dose methotrexate, showed that the null genotype for GSTM1 has not been a prognostic
factor for disease-free survival or probability of hematological remission. However, central
nervous system relapse tended to be less common in those with the GSTM1*0 genotype.
31
32
-3 bp
T-631G
T-567G
C-69T
G-52A
A1*B
M1
A313G
Nucleotide
P1*B
Mutation
I105V
Amino
acid
52
45
55
54
56
42
58
40
40
Caucasian
M3*B
41
27
41
54
African
Frequency
18
2.5
* Clinical effects referred to more than one genotype
Pharmacodynamic
effects
Pharmacokinetic
effects
Phenotype
Table 6: Clinical effects of SNPs in the GST genes
survival for *B/*B genotype in breast cancer patients treated with

cyclophosphamide-containing combination chemotherapy
proportion surviving at 5 years: 66% vs. 86% resp. 0 or 1 *B and
*B/*B
2-fold in risk of relapse in childhood ALL patients with null
genotype
Frequency in ALL patients
Frequency in controls
central nervous system relapse in ALL
At least one allele of GSTM1: trend to a better survival in AML with
doxorubicin treatment. At low accumulated dose *0 survival
survival in adult AML with one GST deletion (M1/T1)*
risk on treatment failure in NHL pediatric patients with at least
one GSTM1 allele
survival in null genotype in lung cancer patients
patients with normal hearing after cisplatin treatment
patients with hearing impairment after cisplatin treatment
predicted CL of etoposide in patients with mutant allele
Clinical effect
[100]
[99]
[97]
[98]
[96]
[95]
[93]
[92]
[39]
Ref
33
A313G
P1*B
T1
Nucleotide
Mutation
I105V
Amino
acid
22
42
2
13
8
32
6
16
30
Caucasian
Frequency
African
Homozygous mutant 3-fold in risk of relapse in childhood ALL

patients
survival of patients with advanced colorectal cancer after
5-FU/oxaliplatin treatment
survival in breast cancer patients
6.7-fold reduction in risk of prednisone poor-response in
children with ALL
5.9-fold reduction in risk of relapse in ALL after prednisone
treatment
Frequency in patients with relapse
Frequency in patients without relapse
2.8-fold in risk of relapse in childhood ALL patients with null
genotype
survival in adult AML with one GST deletion*
Clinical effect
Clinical effects referred to more than one genotype

Abbreviations: AML=Acute Lymphoblastic Leukaemia; AML=Acute Myeloid Leukaemia; CL=clearance; NHL=Non-Hodgkins Lymphoma
Pharmacodynamic
effects
Phenotype
Table 6: Clinical effects of SNPs in the GST genes (continued)
[97]
[93]
[94]
[94]
[103]
[102]
[93]
Ref
Chapter 1.1
In patients with Acute Myeloid Leukemia (AML) receiving doxorubicin induction therapy,
the presence of at least one GSTM1 allele had a trend towards a better survival than patients
with the null genotype. However, at low accumulated doxorubicin dose, the null genotype
had a better survival than patients expressing the gene.[96] In a study of Voso et al.[97] the
appearance of at least one GST deletion (GSTM1 or GSTT1) in adult AML predicted a poor
response and a shorter overall survival after treatment with an anthracycline (doxorubicin,
idarubicin or mitoxantrone), cytarabine and etoposide, followed by chemotherapy
consolidation and stem cell transplantation.
In pediatric non-Hodgkins lymphoma (NHL) patients, treatment failure was significantly less
likely to occur in patients carrying at least one GSTM1 allele in comparison to those with a
homozygous deletion of the GSTM1.[98] Also in lung cancer patients with GSTM1 null
genotype the survival is worse compared with GSTM1 present subjects.[99]
GSTM3
A few effects of polymorphisms in this gene have been observed. The GSTM3 polymorphisms
are in general associated with a variable inducibility of the enzyme. Relatively little is known
about the role of GSTM3 in the metabolism of cytostatic drugs. GSTM3 appears to be critical
in protection against oxidative stress. Cisplatin therapy in malignant neoplasms is associated
with oxidative stress, which possibly results in ototoxicity. In patients treated with cisplatin,
the GSTM3*B allele, had a protective effect. The frequency of this allele in the group with
normal hearing after treatment was 18% versus 2.5% in the group with hearing impairment.[100]
GSTP1
The polymorphisms detected for GSTP1 constitute two variant alleles, GSTP1*B and
GSTP1*C, compared to the wild-type form GSTP1*A (see Table 5). Several anti-neoplastic
drugs used in the neuroblastoma high-risk chemotherapeutic protocol are potential
substrates of GSTP1-1; etoposide, doxorubicin, cisplatin, oxaliplatin and carboplatin (see
Table 2).
The variants decrease GSTP1 activity with some substrates but not with others. See Table 6
for the clinical effects of polymorphisms in the GSTP1 gene. Thus, their actually importance
is not fully understood. Besides, the effects of the genotype of GSTP1 on the
pharmacokinetics of anti-cancer drugs are not studied extensively. Children treated with
etoposide possessing the GSTP1 wild type genotype showed a significantly lower clearance
of etoposide.[39] The homozygous GSTP1*B genotype showed a 3-fold increase in the risk of
relapse with childhood ALL patients.[93] No GSTP1 polymorphism had an impact on patient
response to the treatment of neuroblastoma.[101] Patients with colorectal cancer and treated
with 5-FU/oxaliplatin showed an increased survival if the mutant GSTP1*B allele was
present.[102] Also in breast cancer patients a better survival was observed for the GSTP1*B
genotype.[103] Because of the presence of less active GSTP1, the elimination rate is reduced
and a prolonged exposure of the drug to the tumour is achieved. Thus a better survival could
be expected.
34

GSTT1
A homozygous deletion of the entire GSTT1 gene (null genotype or *0) has been associated
with lack of GSTT1 activity.
A correlation between GST polymorphisms and prednisone response in children with ALL
has been found. The homozygous deletion of GSTT1 conferred a 6.7-fold reduction in risk of
prednisone poor-response. In addition, risk of relapse was predicted strongest by the GSTT1
genotype, the null genotype conferred a 5.9 fold reduction in risk of relapse.[94] The GSTT1*0
genotype conferred a 2.8-fold reduced risk of relapse in childhood ALL patients.[93] In
contrast, the appearance of a GSTT1 deletion in adult AML proved to be a poor prognostic
factor for survival.[97]
Overall, few data are available for the correlation between the genotype of GST and
pharmacokinetic parameters for anti-cancer drugs. For pharmacodynamic correlations more
studies have been performed, but data are not consistent. For example, GSTM1*0 and
GSTT1*0 reduced risk of relapse in ALL, but also reduced survival in AML patients. Further
research has to be done to evaluate the influence of polymorphisms on the efficacy and
toxicity of specific anti-cancer drugs separately.
Sulfotranspherase (SULT)
SULT1A1
Carlini et al.[104] reported a wide racial variation in SULT1A1 polymorphisms. SULT1A1*2 (see
Table 5) results in significantly reduced enzyme activity and thermo stability. Allele
frequencies for SULT1A1*1 and *2 in Caucasian subjects were 65.6 and 33.2, respectively.
Frequencies for those same alleles were different for Chinese subjects: 91.4 and 8.0,
respectively as were frequencies in African-American subjects: 47.7 and 29.4%, respectively.
In an Egyptian population the allele frequency of *2 was 13.5%.[105]
No effects of polymorphisms in the SULT1A1 gene on the pharmacokinetics of anti-cancer
drugs have been reported yet. In a study of Nowell et al.[106] the influence of the SULT1A1
polymorphism on the efficacy and toxicity of anti-cancer treatment has been studied.
Homozygosity of the SULT1A1*2 allele has been associated with three times the risk of death
after tamoxifen treatment in breast cancer patients For other anti-cancer drugs and tumour
types no association can be made between the genotype of SULT1A1 and the
pharmacokinetics and pharmacodynamics of the drugs.
Arylamine N-Acetyltransferase (NAT)

Two NAT iso-enzymes have been identified in humans, namely NAT1 and NAT2, which are
products of distinct genetic loci, designated NAT1 and NAT2, respectively. The two
functional NAT genes share 87% nucleotide identity, which translates to an 81% homology at
the amino acid level. Both NAT1 and NAT2 genes display pronounced allelic variation, with
35
Chapter 1.1
26 different human NAT1 and 29 different human NAT2 alleles identified to date
(www.louisville.edu/medschool/pharmacology/NAT.html).[107] Only a small number have
been shown to alter phenotype in vivo. A population study of Butcher et al.[108] showed a
bimodal distribution of NAT1 activity, with 8% of the individuals being slow acetylators.
Moreover, the above study was one of the first to report a correlation between NAT1*14 and
NAT1*17 genotype and slow acetylator phenotype (see Table 5). However, the frequency of
slow acetylator alleles for NAT1 is low. The occurrence of the most common low activity
allele, NAT1*14, ranges from 1.3 to 3.7% in a Caucasian population.[107] A much higher
frequency was found in a Lebanese population, 50% of the population had a slow acetylator
genotype.[109] This indicates that considerable interethnic variability exists in frequencies of
polymorphisms in the NAT1 gene.
Of the 29 different NAT2 genes detected in human populations, each is comprised of one to
four nucleotide substitutions. Thirteen of these nucleotide substitutions have been identified
within the 870-bp coding region of the gene. Polymorphisms leading to a slow acetylator
phenotype are: NAT2*5, *6, *7, *10, *14, *17, *18 and *19 (see Table 5). NAT2 shows more
marked interethnic variability of allelic variants than NAT1.[110]
Several studies have been performed that show clear correlations between NAT2 genotype
and phenotype.[111-113] A gene-dosage effect has been identified, as individuals homozygous
for NAT2 polymorphisms had a slow acetylator phenotype, individuals heterozygous for
NAT2 polymorphisms had an intermediate acetylator phenotype and individuals who lacked
polymorphisms (NAT2*4) had a rapid acetylator phenotype.
Both enzymes catalyze N-acetylation (usually deactivation) and O-acetylation (usually
activation) of aromatic and heterocyclic amine carcinogens. Amonafide maleaat is a novel
arylamine that has previously been used in clinical trials for the treatment of various cancers.
It undergoes N-acetylation to an active metabolite that contributes to systemic toxicity.
Several studies have shown that myelosuppression is greater in rapid acetylators compared
to slow acetylators following a standard dose of amonafide.[114-116] This has led to different
recommended doses for the two groups. No further research has been performed on
pharmacokinetics and clinical outcome of anticancer drugs associated with polymorphisms
in the NAT gene.
Recent reports suggesting that NAT activity may be altered by environmental factors and
substrate-dependent down-regulation, such as paclitaxel, may also explain why inconsistent
associations have been reported.[117,118]
Thiopurine methyltransferase (TPMT)

Thiopurine methyltransferase (TPMT) is involved in the methylation reactions of 6mercaptopurine (6-MP) and azathioprine, its pro-drug.[119] Since 1953, 6-MP has been
administered in the treatment of childhood ALL. More experience with this drug and
azathioprine has been obtained in the treatment of rheumatoid arthritis and inflammatory
bowl diseases.
36

The activity of TPMT is influenced by the genetic polymorphisms which can alter the rate of
6-MP metabolism by TPMT (see Table 5). The enzyme activity of TPMT varies among
patients; 86.6% of the Caucasian population has high TPMT activity, 11.1% has intermediate
activity, and 0.3% are deficient in TPMT.[120-122] The TPMT found in patients with normal TPMT
activity is classified as the wild-type TPMT*1.
Eight TPMT alleles have been identified. Three of these alleles (TPMT*2,[123] *3A and *3C
(G460A) see Table 5) account for 80-95% of intermediate- or low-enzyme activity cases.[120122,124-128]
High concentrations of variant TPMT (TPMT*3A, *3B and *3C) are found in patients
with decreased TPMT activity: patients with TMPT-3A have a complete loss of TPMT catalytic
activity, patients with TPMT*3B have a nine-fold reduction, and those with TPMT*3C have a
1.4 fold reduction.[121,124] The frequency of loss of TPMT activity (TPMT*3A) appears to vary
with ethnicity, but not with gender or age.[120-122,125,129,130]
Polymorphisms have also been described within the 5-flanking promoter region of the TPMT
gene due to a variable number of tandem repeats (VNTR*3-*8),[131,132] however TPMT
promoter VNTRs are unlikely to play a significant role in changes in TPMT activity in
response to azathioprine therapy.[133-136] Recently, novel missense mutations have been
described: M1V (TPMT*14 ), IVS7-1G>A (TPMT*15),[137] L122T (TPMT*19),[138] R163H
(TPMT*16), K119T, Q42E and G71R.[139] Taking these new variants into consideration, the
overall concordance rate between TPMT genetics and phenotypes was 98% in a European
Caucasian population.[130,139] In addition, haplotyping methods have been developed to
discriminate the genotypes TPMT*1/*3A (intermediate metaboliser) and TPMT*3B/*3C
(poor metaboliser).[140]
The TPMT genotype correlates well with in vivo enzyme activity within erythrocytes and
leukaemic blast cells and is clearly associated with risk of toxicity.[122,141,142] The cumulative
incidence of 6-MP dose reduction due to toxicity was highest among patients homozygous
mutant for TPMT, intermediate among heterozygous patients and lowest among wild-type
patients.[143,144] Unfortunately, in the published studies often no distinction has been made
between the different polymorphisms of the TPMT gene. The concentration of 6-thioguanine
nucleotides (6-TG) in erythrocytes is directly correlated with risk of development of
leukopenia and inversely with risk of relapse in patients treated with thiopurine drugs. Dose
reduction in patients with inactive TPMT is associated with lower 6-TG concentrations and
thus a higher risk or relapse.[145] The TPMT genotype has a substantial impact on minimal
residual disease after administration of 6-MP in the early cours of childhood ALL.[146] In
addition, there are emerging data that the TPMT genotype may influence the risk of
secondary malignancies, including brain tumours and AML. High dose 6-MP in wild-type
patients with ALL has been associated with a higher occurrence of infectious episodes,
which could not be influenced by dose adjustment in homozygous and heterozygous mutant
patients during maintenance therapy.[147]
Tests for the TPMT genotype and phenotype are commercially available. Attention should be
paid for those patients tested negative for TPMT status. Patients with poor or intermediate
TPMT activity may tolerate only 1/10 to 1/2 of the average 6-MP dose. A pharmacoeconomic
37
Chapter 1.1
model has been developed to analyse the potential cost of screening to prevent azathioprine
toxicity. In this model it is assumed that TPMT deficiency is present in 0.3% of the population
and intermediate activity is present in 11%, and that both groups have an increased risk of
developing myelosuppression. Under these circumstances the model predicted that the costs
per Caucasian patient for the first 6 months of therapy with screening are lower compared to
not screening.[148,149] In addition, the genotype-based dosing strategy was also shown to be less
costly and more effective in Korea, as screening was associated with a marked reduction in
the number of serious adverse events.[150]
Overall, genetic variation in the TPMT gene has clearly shown its importance for the clinical
response in anti-cancer treatment in ALL. This not only results in a clinical benefit for the
patient, but also in a significant impact on the economics of medical practice.
Drug transporters
P-glycoprotein (P-gp)
P-glycoprotein (P-gp, MDR1, ABCB1) consists of two similar halves, each containing six
putative transmembrane regions and an ATP binding/utilization domain, separated by a
flexible linker polypeptide.[151] ATP binding and hydrolysis appears to be essential for the
proper functioning of P-gp, including drug transport. P-gp is a member of the ATP binding
cassette (ABC) family of transporters. Substrates for P-gp cover a variety of chemical
structures, and have diverse therapeutic indications. The molecules are usually lipophilic
and amphipathic, containing one or more aromatic rings. P-gp has been implicated in
multidrug resistance (MDR), which is a major problem in cancer.
The bioavailability of drugs will be limited by P-gp for substrates that have a high affinity for
P-gp and are applied orally. Some anti-cancer drugs that are substrate for P-gp are mentioned
in Table 2.
Genetic polymorphisms in the P-gp gene may be important in influencing the outcome of
pharmacotherapy,[152] and several SNPs in the ABCB1 gene have been described. The three
most mentioned SNPs are P-gp*6, *7 and *8 (see Table 7).
In Caucasian populations a frequency of 22-56% for the mutant T allele in P-gp*6 was
observed.[153-155] In African populations the frequency of the C allele was significantly higher in
comparison with Caucasians (respectively 80% and 50%).[156]
A common haplotype was found to contain three SNPs simultaneously (P-gp*2): *6, *7 and
*8, with a frequency of 62% among European Americans.[157] Direct sequencing of DNA from
subjects homozygous for all of these 3 SNPs strongly suggested that they were linked to
polymorphic positions at regulatory sites of the P-gp promoter and may account for different
regulatory kinetics.
For P-gp*6, a significant correlation has been observed between P-gp expression levels and
function of P-gp. Individuals homozygous mutant for *6 had a significantly lower duodenal
P-gp expression and an increase in digoxin plasma levels after oral administration of
digoxin.[155] In contrast to these findings, Sakaeda et al.[158] reported a higher exposure of
38

digoxin after a single oral dose in Japanese subjects with the wild-type *6 genotype
compared to heterozygous and homozygous mutants. The reason for those apparent
discrepancies is unknown at the moment.
Table 7: Characteristics of drug transporter genes (with the most important
polymorphisms)
Gene
Location
(chromosome)
Protein
Exons
Amino
acids
ABCB1
7q21
P-gp
MDR1
ABCC2
10q24
MRP2
32
1545
ABCG2
4q22
BCRP
16
655
29
1280
Polymorphisms
Location
(Exon)
Effect
*6 (C3435T)
*7 (G2677T/A)
*8 (C1236T)
G1199A
C-24T
C1249A
C2302T
C2366T
T2439+2C
?
C3972T
A4145G
G4348A
G34A
C8825A
26
21
12
11
5-UTR
10
18
18
18
26
28
29
31
2
5
A893S
S400N
V417I
R768W
S789F
Splice site
W1254Y,A,C,F
Q1382R
G1440S
V12M
Q141K
Recently, the G1199A polymorphism in P-gp has been studied. This polymorphism alters the
efflux and transepithelial permeability of rhodamine-123. Cells with the G1199A
polymorphism were more resistant to vinblastine and vincristine. No difference in resistance
was found for doxorubicin.[159] Thus, this polymorphism may influence drug disposition and
therapeutic efficacy of vinblastine and vincristine. However, in an in vivo study of
Plasschaert et al.[160] no association was observed between *6 or *7 and vincristine
pharmacokinetics variables. When haplotypes were assigned, a correlation was identified
between the longer elimination half-life and wild-type alleles of *6 and *7. Haplotypes of
these polymorphisms did not affect other pharmacokinetics parameters, such as AUC and
clearance, suggesting that the observed effect on elimination half-life is of very limited
relevance.
P-gp is responsible for transport of the carboxylate form of irinotecan.[161] The homozygous
mutant *8 polymorphism has been associated with significantly increased exposure to
irinotecan and its active metabolite SN-38.[162] Also a trend to an increased AUC of tipifarnib
in patients with the homozygous mutant allele compared to patients with only one or no
mutant alleles of *8, has been found in a study of Sparreboom et al.[163] (See Table 8)
Goh et al.[164] studied the effects of polymorphisms in the P-gp gene on the pharmacokinetics
of docetaxel. There were no significant differences between clearances of docetaxel or
midazolam according to P-gp genotype. However, the two patients with the lowest and
highest docetaxel clearances had homozygous mutant *6 and wild-type genotypes,
39
40
C1236T
*8
C3435T
*6
G2677T
C3435T
*6
*7
C1236T
Nucleotide
*8
Mutation
Silent
A893S
Silent
Silent
Silent
Amino
acid
[165]
[166]
[165]
Wild-type overall survival and high probability of relapse in AML

risk of drug resistance in treatment for lymphoproliferative diseases
41.7
40.5
[165]
[166]

risk of drug resistance in treatment for lymphoproliferative diseases in
mutant alleles
response in preoperative chemotherapy in breast cancer patients in
mutant alleles
49.6
[167]
[39]
predicted CL of etoposide
Ref
[162]
[163]
Clinical effect
exposure of irinotecan and SN-38 for TT alleles
Trend for AUC of tipifarnib
Caucasian
Frequency
Abbreviations: AML=Acute Myeloid Leukaemia; AUC= area under the concentration-time curve; CL=clearance
Pharmacodynamic
effects
Pharmacokinetic
effects
Phenotype
Table 8: Effects of SNPs in the P-gp gene

respectively. In a study of Kishi et al.[39] the mutant allele for *6 was also correlated with a
lower clearance of etoposide in children with ALL.
P-gp gene polymorphisms are also reported to affect the outcome of therapy in patients with
AML. Illmer et al.[165] compared the clinical course of AML treatment with the anti-cancer
drugs etoposide, mitoxantrone or daunorubicin among patients with various P-gp genotypes,
and demonstrated that patients homozygous for the wild type allele at any locus investigated
(exons 12, 21 and 26) exhibited a significantly decreased overall survival with a higher
probability of relapse. Theoretically, a reduced intracellular concentration of anti-cancer
drugs attribuTable to the action of P-gp in AML blasts may be related to failure of AML
therapy and disease resistance. Illmer et al.[165] did not determine whether the association
between P-gp polymorphism and survival of AML patients was attribuTable to altered P-gpmediated drug pharmacokinetics. However, a clear correlation between homozygosity for
the wild type allele(s) and lower P-gp expression in blast samples was detected. In a study of
Goreva et al.[166] individuals with homozygous mutations for *6 and *7 are at highest risk of
drug resistance in the treatment for lymphoproliferative diseases. Another study showed that
the response to preoperative chemotherapy in breast cancer patients was higher in patients
with the homozygous mutant *6 genotype.[167] Efferth et al.[168] did not find a difference in
overall survival for wild type and mutant *6 genotype alleles in ALL after doxorubicin
treatment.
In conclusion, contadictory data have been published for P-gp genotypes. The studies
discussed are evaluating different regimens and tumour types. The same protein can have
different pharmacokinetic and pharmacodynamic effects in different tumours and in
response to different drugs. Therefore, every tymour type and drug has to be investigated
independently.
MRP2 (canalicular-multispecific organic anion transporter cMOAT)

The multidrug resistance associated protein 2 (MRP2; ABCC2, also termed canalicular
multispecific organic anion transporter (cMOAT)) is an ATP-binding cassette transporter
accepting a different range of substrates, including glutathione, glucuronide, and sulphate
conjugates of many endo- and xenobiotics, and is involved in the excretion of these
conjugates into the bile.[169] MRP2 generally performs excretory or protective roles.[170]
MRP1 and MRP2 have a five domain structure in common with a core consisting of two
tandemly arranged units, each containing a membrane spanning domain (MSD) and a
nucleotide-binding domain (NBD) that is preceded by a third MSD with an extracytosolic
NH2 terminus. Although the amino acid similarity between MRP1 and MRP2 is only 49%, both
ABC transporters share similar substrate specificity. The transport or recognition of substrates
41
Chapter 1.1
for MRP2 may be dependent on the charged amino acids in the transmembrane domain of
MRP2, as has been shown in a study of Ito et al.[171]
MRP2 mediates among others hepatobiliary excretion of conjugated bilirubin. Alteration of
this transport system causes Dubin-Johnson syndrome (DJS), which is a hereditary disease
characterized by conjugated hyperbilirubinaemia and an increase in the urinary excretion of
co-proporphyrin isomer I (a metabolic byproduct of heme synthesis and a substrate for
MRP2).[170]
In the MRP2/cMOAT gene, mutations have been observed that are responsible for DJS.[172-175]
The frequencies of all these polymorphisms have not been studied in Caucasian subjects.
Studies in Japanese and Jewish subjects were performed, but only few subjects were
included.[172,173,176-180] In most studies all subjects having mutations in the MRP2 gene were
heterozygous carriers. Toh et al.[173] reported that heterozygous carriers showed comparable
serum bilirubin concentrations but higher urinary levels of coproporphyrin isomer I than
normal, and suggested a correlation between the urinary level of coproporphyrin isomer I
and the homozygous/heterozygous status of mutation in the MRP2 gene.
Anti-cancer drugs that are substrate for MRP2 include doxorubicin, vinblastine,
sulfinpyrazone, irinotecan, SN38 and methotrexate (see Table 2). The transport and/or
pharmacokinetics of these substrates may be affected by mutations in the MRP2 gene,
however it is currently unknown whether mutations in the MRP2 gene have consequences
for the activity or toxicity of these anti-cancer drugs.[173]
An amino acid change of W1254Y or A or C or F eliminated transport of methotrexate.[181] The
tryptophan at position 1254 in the putative last transmembrane segment plays a critical role
in the ability of MRP2 to transport methotrexate. However, this mutation was studied in cell
lines and it is not known what influence this mutation has on transport in cells in vivo.
The protein levels of MRP2 did not correlate with decreased sensitivity of lung cancer cell
lines (NSCLC) to doxorubicin, vincristine, etoposide, and cisplatin, as apposed to MRP1 and
MRP3.[182] MRP2 expression was increased by >10 fold in renal brush-border membranes in
rats after cisplatin administration, although it had no effect on liver MRP2 expression in
rats.[183] In other studies, the expression of MRP2 in rat hepatocytes increased after cisplatin
administration in a study of Kauffmann et al.[184] and in HEPG2 cells cisplatin also induces the
expression of MRP2.[185] Treatment with tamoxifen in rhesus monkeys (male and female)
resulted in a strong increase in MRP2 mRNA in the liver.[186] This may be of significant
relevance for the acquisition of multidrug resistance during chemotherapy.
MRP2 mRNA expression was low in normal colorectal mucosa and specifically increased in
cancer regions compared with noncancerous regions. The mRNA expression of MRP2 in
these tissues was significantly associated with resistance to cisplatin.[187] In humans the MRP2
expression in recurrent and residual tumours of the bladder after chemotherapeutic
treatment with doxorubicin was higher than that in untreated primary tumours.[188] In a study
of Burger et al.[189] no significant association of expression levels of MRP2 and length of
progression free survival and post-relapse overall survival could be detected. Progression-free
42

survival in ovarian carcinoma patients showed a clear tendency to be associated with no
expression for MRP2.[190]
Overall, no clinical effects have been observed for the different MRP2 genotypes and
pharmacokinetics and pharmacodynamics of anti-cancer drugs. A possible explanation
could be in the presence of a lesser active MRP2 transporter, due to a overlap of substrate
specificity (see Table 2), other transporters, such as P-gp, BCRP or MRP4, will take over the
role of drug transporter.
Breast Cancer Resistance Protein (BCRP)

The ABCG2 gene is a recently described member of the G subfamily of ABC transporters, and
was previously called Breast Cancer Resistant Protein (BCRP), Mitoxantrone Resistant
Protein (MXR) or Placenta-specific ATP binding cassette transporter (ABCP).[191-194]
The protein with six transmembrane segments and one ATP-binding cassette is located in the
plasma membrane.[195] Recently Xu et al.[196] found that human ABCG2 exists mainly as a
homo-tetramer with a possibility of a higher form of oligomerization consisting of 12
subunits.
The role of ABCG2 seems to be protecting tissues by actively transporting toxic substances
and xenobiotics out of the cells. Cancer cells over expressing ABCG2 show multidrug
resistance to mitoxantrone, methotrexate, doxorubicin and camptothecin-based anti-cancer
drugs, such as topotecan and SN-38 (see Table 2). Large interindividual differences have
been observed in oral availability and clearance of drugs that are substrate for ABCG2,
especially topotecan.[197] Genetic variation in the ABCG2 gene could possibly explain the
variability in pharmacokinetics of ABCG2 substrates. SNPs have already been reported in the
ABCG2 gene.[194,198-202]
Mizuarai et al.[203] analysed the effect of the polymorphisms G34A and C8825A, leading to an
amino acid change of V12M and Q141K respectively (see Table 7), on the transporter
function of the protein. Drug resistance to indolocarbazole, a topoisomerase I inhibitor, of
cells expressing V12M or Q141K was less then 1/10 compared to wild type ABCG2 transfected
cells and was accompanied by increased drug accumulation and decreased drug efflux in
the variant ABCG2 expressing cells. A possible explanation for this altered function of the
ABCG2 enzyme is the fact that the ABCG2 transporter is not localized to the apical
membrane in the V12M clone. However, it is not known if the altered transport function of
ABCG2 influences drug transport in vivo.
In a study of Imai et al.[198] Japanese volunteers with the mutant C8825A polymorphism (allele
frequency of 46%) express low amount of the Q141K ABCG2 protein. In the cancer cell line
PA317, this SNP showed a markedly decreased protein expression and low-level drug
resistance, which can result in hypersensitivity of normal cells to anti-cancer drugs. The
pharmacokinetics of diflomotecan have been affected by this polymorphism.[204] Patients
heterozygous for this allele showed statistically significant increased plasma levels in
comparison to patients with wild-type alleles. De Jong et al.[205] however, showed that no
significant changes in irinotecan pharmacokinetics in vivo could be observed in relation to
43
Chapter 1.1
the ABCG2 C8825A genotype, although one of two homozygous variant allele carriers
showed extensive accumulation of SN-38 and SN-38G.
In Acute Myeloid Leukaemia (AML) cells, ABCG2 is over expressed.[206-210] A few clinical
studies reported that some AML and breast cancers expressed ABCG2 at a mRNA or protein
levels that can be detected,[207,209,211-213] however the association between the expression and
clinical resistance to anti-cancer drugs remains undetermined. Van der Kolk et al.[213] did not
find a relation between the up-regulation of ABCG2 and relapsed or refractory AML.
The levels of ABCG2 mRNA expression in cell lines were found to be significantly correlated
with the ABCG2 function and the sensitivity to SN-38 and topotecan.[214] In Non Small Cell
Lung Cancer (NSCLC) tissues, the ABCG2 mRNA expression levels were widely dispersed.
Five (22%) out of 23 tissues expressed higher levels of the ABCG2 mRNA compared to the
ABCG2 mRNA levels in NCI-H441 cells with active ABCG2 function conferring high resistance
to topotecan in vitro.[214]
The identification of functional sequence variation in the ABCG2 gene could also be of
interest in the field of prognosis of disease. High expression levels of ABCG2 have been
associated with a worse clinical outcome in AML.[210] Recently Yoh et al.[215] concluded that
positive immunostaining for ABCG2 appears to be a negative predictor of survival in patients
with advanced NSCLC. The response rate was lower in ABCG2-positive tumours, although not
significantly. ABCG2-positive patients had also shorter progression-free survival and overall
survival than ABCG2-negative patients. In other studies no proof of over expression was
determined in breast and lung cancer.[191,211,216,217]
In conclusion, to date no in vivo effects of BCRP polymorphisms have been detected in
relation to efficacy and toxicity of anti-cancer drugs that are substrates for BCRP.
Influx transporters
Influx transporters are increasingly recognised as important for drug action, and the genetics
of these influx transporters are currently being investigated. For example, the organic anion
transporter polypeptide-1B1 (OATP-1B1, SLCO1B1) is involved in the liver uptake of several
compounds. Innocenti et al.[218] showed that patients with heterozygous or homozygous
SLCO1B1*5 (T521C) genotype had a higher irinotecan AUC and that SLCO1B1*1b (A388G)
was associated with a more severe neutropenia after irinotecan treatment. Polymorphisms in
these kind of genes can possibly contribute in the explanation of the interindividual
variability in anti-cancer therapy. In the future, research will be focused on polymorphisms
in all kinds of genes responsible for the uptake, activation, degradation and excretion of anticancer drugs.
Conclusions
A number of polymorphisms have been described in genes coding for drug metabolising
enzymes and drug transporters. However, in most cases no clear association has been found
for polymorphisms in these enzymes and pharmacokinetics or pharmacodynamics of anticancer drugs. The polymorphisms have been identified in relatively small studies. In other
44

cases no pharmacogenetic studies have been published at all. For cyclophosphamide and
ifosfamide, the influence of polymorphosms in the CYP2B6 and ALDH genes has not been
investigated even though the metabolism of these anti-cancer drugs by these enzymes ia
known. To examine the influence of these polymorphisms in more detail, larger studies and
possibly pooling data from different studies may be necessary. But using different genotyping
techniques and using patients from different ethnic origin, that often show different
frequencies for mutant alleles, makes it difficult to compare the different studies. Recent
advances in gene expression array technology, high-throughput genotyping, and
bioinformatics have greatly contributed to the expansion in pharmacogenetic research
methodology.
Any association between polymorphisms in genes for metabolising enzymes and drug
transporters and efficacy or toxicity of an anti-cancer treatment could result from linkage
disequilibrium (LD). LD arises when a specific allele is located a short distance from another
gene variant on the same chromosome that actually cause the variability in efficacy or
toxicity. Molecular haplotyping techniques and analysis of linkages on the same
chromosome directly by biophysical and biochemical means, may be needed to
characterize haplotypes in individuals with a highly polymorphic and large gene. Haplotype
identification may prove to be vital in identifying the functional significance of
polymorphisms on drug metabolism and disposition.[219]
The promise of pharmacogenetics is tailored drug therapy, in which SNPs are identified that
are associated with life-threatening adverse events or low efficacy of therapy. With validated
SNPs and haplotype markers that are associated with toxicity and efficacy, clinicians could
perform screening tests and, if indicated, choose alternate dosages or therapy. The clinician
will be faced with assessing the overall risk for adverse effects, which have to be weighed
against potential benefits as well as the availability of alternative therapies and the costs. The
reality is that clinicians will have to make their decisions based on the risk assessment
gathered from large, population-based studies. However, this information is not available at
this moment. Detailed knowledge of the scope and pattern of the genetic information will be
required, not only from SNPs, but also of haplotypes and LD for genes and pathways critical
to cancer therapy in different populations.
Currently, only one specific recommendation can be made to optimize anti-cancer therapy
for patients by genotyping drug metabolising enzymes and drug transporters: i.e. in patients
with a reduced activity of TPMT (based on pharmacogenetic screening of the TPMT gene or
activity analysis), the dose of 6-MP should be reduced to 1/2 or 1/10. The most promising
polymorphisms for pretreatment screening in the future are the UGT1A1*28 and mutations in
the DPYD gene, because pharmacogenetic research has shown significant relationships
between pharmacokinetic variability in these genes on the one hand and drug-related
toxicity and variability in tumour reponse on the other. Well-designed and well-executed
45
Chapter 1.1
studies will help demonstrate convincing links between genetic variation and drug toxicity
and responses.
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MRP2/cMOAT/ABCC2 causes Dubin-Johnson syndrome in a Caucasian patient. J Hum Genet
2003;48(9):484-486
181. Ito K, Oleschuk CJ, Westlake C et al. Mutation of Trp1254 in the multispecific organic anion
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183. Demeule M, Brossard M, Beliveau R. Cisplatin induces renal expression of P-glycoprotein and
canalicular multispecific organic anion transporter. Am J Physiol 1999;277(6 Pt 2):F832-F840
184. Kauffmann HM, Keppler D, Kartenbeck J et al. Induction of cMrp/cMoat gene expression by
cisplatin, 2-acetylaminofluorene, or cycloheximide in rat hepatocytes. Hepatology
1997;26(4):980-985
185. Schrenk D, Baus PR, Ermel N et al. Up-regulation of transporters of the MRP family by drugs
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Hinoshita E, Uchiumi T, Taguchi K et al. Increased expression of an ATP-binding cassette
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Tada Y, Wada M, Migita T et al. Increased expression of multidrug resistance-associated
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Burger H, Foekens JA, Look MP et al. RNA expression of breast cancer resistance protein, lung
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Allen JD, Schinkel AH. Multidrug resistance and pharmacological protection mediated by the
breast cancer resistance protein (BCRP/ABCG2). Mol Cancer Ther 2002;1(6):427-434
Zamber CP, Lamba JK, Yasuda K et al. Natural allelic variants of breast cancer resistance
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Mitomo H, Kato R, Ito A et al. A functional study on polymorphism of the ATP-binding
cassette transporter ABCG2: critical role of arginine-482 in methotrexate transport. Biochem J
2003;373(Pt 3):767-774
Xu J, Liu Y, Yang Y et al. Characterization of oligomeric human half-ABC transporter ATPbinding cassette G2. J Biol Chem 2004;279(19):19781-19789
Kruijtzer CM, Beijnen JH, Rosing H et al. Increased oral bioavailability of topotecan in
combination with the breast cancer resistance protein and P-glycoprotein inhibitor GF120918.
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Imai Y, Nakane M, Kage K et al. C421A polymorphism in the human breast cancer resistance
protein gene is associated with low expression of Q141K protein and low-level drug
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Backstrom G, Taipalensuu J, Melhus H et al. Genetic variation in the ATP-binding Cassette
Transporter gene ABCG2 (BCRP) in a Swedish population. Eur J Pharm Sci 2003;18(5):359-364
Iida A, Saito S, Sekine A et al. Catalog of 605 single-nucleotide polymorphisms (SNPs) among
13 genes encoding human ATP-binding cassette transporters: ABCA4, ABCA7, ABCA8, ABCD1,
ABCD3, ABCD4, ABCE1, ABCF1, ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8. J Hum Genet
2002;47(6):285-310
Honjo Y, Morisaki K, Huff LM et al. Single-nucleotide polymorphism (SNP) analysis in the ABC
half-transporter ABCG2 (MXR/BCRP/ABCP1). Cancer Biol Ther 2002;1(6):696-702
Bosch TM, Kjellberg LM, Bouwers A et al. Detection of SNPs in the ABCG2 gene in a Dutch
population. Am J Pharmacogenomics 2005;5(2):123-131
Mizuarai S, Aozasa N, Kotani H. Single nucleotide polymorphisms result in impaired
membrane localization and reduced atpase activity in multidrug transporter ABCG2. Int J
Cancer 2004;109(2):238-246
Sparreboom A, Gelderblom H, Marsh S et al. Diflomotecan pharmacokinetics in relation to
ABCG2 421C>A genotype. Clin Pharmacol Ther 2004;76(1):38-44
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protein messenger RNA expression in mitoxantrone-selected cell lines. J Natl Cancer Inst
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Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent
transporters. Nat Rev Cancer 2002;2(1):48-58
Sargent JM, Williamson CJ, Maliepaard M et al. Breast cancer resistance protein expression
and resistance to daunorubicin in blast cells from patients with acute myeloid leukaemia. Br J
Haematol 2001;115(2):257-262
van den Heuvel-Eibrink MM, Wiemer EA, Prins A et al. Increased expression of the breast
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Steinbach D, Sell W, Voigt A et al. BCRP gene expression is associated with a poor response to
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Faneyte IF, Kristel PM, Maliepaard M et al. Expression of the breast cancer resistance protein
in breast cancer. Clin Cancer Res 2002;8(4):1068-1074
Ross DD, Karp JE, Chen TT et al. Expression of breast cancer resistance protein in blast cells
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and murine lung. J Clin Pathol 2002;55(5):332-339
Innocenti F, Undevia SD, Rosner GL et al. Irinotecan (CPT-11) pharmacokinetics (PK) and
neutropenia: interaction among UGT1A1 and transporter genes. ASCO 2005;2006
Woodahl EL, Ho RJ. The role of MDR1 genetic polymorphisms in interindividual variability in
P-glycoprotein expression and function. Curr Drug Metab 2004;5(1):11-19
Chapter 1.2
Genetic polymorphisms of drug
metabolising enzymes and drug transporters
in relation to cancer risk
Tessa M. Bosch
Irma Meijerman
Jos H. Beijnen
Jan H.M. Schellens
Submitted
Chapter 1.2
Abstract
There is a wide variation in cancer incidence in humans, which, in part, has been attributed
to metabolic factors of carcinogens and genetic polymorphisms in drug metabolising
enzymes and drug transporters. Drug metabolising enzymes are responsible for the initial
activation of many (pro)carcinogens, such as polycyclic aromatic hydrocarbons (PAH), to
biologically reactive metabolites. Besides, detoxifying enzymes are responsible for the
inactivation of these active carcinogens and deficiency of these enzymes may result in an
increase of cancer risk in exposed individuals. Another factor influencing interindividual
variability in cancer incidence are transporters, which are responsible for the excretion of
carcinogens. A high number of polymorphisms have been described in drug metabolising
enzymes and drug transporter genes. These polymorphisms might influence the activity of
metabolising enzymes and drug transporters and thereby affect cancer risk. This review will
focus on the role of genetic polymorphisms of selected drug metabolising enzymes (CYP1A1,
2C9, 2C19, 3A4, 3A5, UGT1A1, GSTM1, GSTP1, GSTT1, SULT1A1, NAT1 and NAT2) and ABCtransporters (P-gp and BRCP) in relation to cancer risk.
60
Introduction
Environmental factors such as smoking cigarettes, diets and alcohol may interact with
genetic factors, which might results in an individual at a greater or lesser risk of developing a
particular cancer than another. Individuals with heavy exposure, such as smokers and
exposed workers, and individuals carrying cancer-predetermining germ-line mutations in
high-penetrance genes such as BRCA1 and BRCA2, confer a very high risk for cancer. There
is also another group of predisposing polymorphic, low-penetrance genes, i.e. those involved
in carcinogen metabolism, transport and DNA repair, which modestly increase the risk for
cancer in exposed individuals, perhaps already at low doses of carcinogens. In the latter
case, the proportion of cancers attributable to such genetic traits may be high, because the
frequency of at risk alleles in the population is high.[1]
Drug metabolising enzymes may be linked with the initial activation of many
(pro)carcinogens, such as polycyclic aromatic hydrocarbons (PAH), to biologically reactive
metabolites. For example, CYP1A2 activates aromatic and heterocyclic amines from tobacco
and dietary procarcinogens.[2] Binding of reactive metabolites to DNA can result in altered
nucleotide sequences in regulatory or coding regions of genes which, in turn, may affect
gene expressions or functions of resulting transcript or proteins. Besides, deficiency of
detoxifying enzymes may affect the metabolic fates of these chemicals and also raise cancer
risks in exposed individuals.
Advances in molecular biology have allowed the identification of many allelic variants of
several drug metabolising enzymes and drug transporters so that individuals with susceptible
genotypes can be determined more easily. Several polymorphisms that cause enzyme
deficiencies have been detected in metabolising enzymes, thus can give a higher risk of
cancer. CYP3A4 is involved in the oxidation of testosterone to 2-, 6- and 15-hydroxy
testosterone. A decreased oxidation, caused by polymorphisms in the CYP3A4 gene,
increases the conversion to dihydrotestosterone, which is involved in the regulation of
prostate cell growth and function.[3] Also polymorphisms in transporter genes can influence
the excretion of carcinogens, and thereby the risk of cancer.[4]
Several reviews have been published about this subject, but only in a specific cancer or for
specific genes.[5,6] This review will focus on the role of genetic polymorphisms of several
selected drug-metabolising enzymes (CYP1A1, 1A2, 2C9, 2C19, 3A4, 3A5, UGT1A1, GSTM1,
GSTP1, GSTT1, SULT1A1, NAT1 and NAT2) and ABC-transporters (P-gp and BCRP) and their
possible influence on cancer risk.
Phase I enzymes
Cytochrome P450 (CYP)
CYP1A1
The human CYP1A1 enzyme, present in many epithelial tissues and well conserved, is
involved in the activation of major classes of tobacco procarcinogens, like polycyclic
61
Chapter 1.2
aromatic hydrocarbons (PAH) and aromatic amines. CYP1A1 is also involved in the
metabolism of oestrogen, which is converted into the carcinogenic catecholoestrogens.
The CYP1A1 gene is located on chromosome 15q24 and the most described Single
Nucleotide Polymorphisms (SNPs) are CYP1A1*2A (a T3801C change, also named m1),
CYP1A1*2C (A2455G or m2, in exon 7 causing an amino acid change of I462V), CYP1A1*3
(T3205C or m3) and CYP1A1*4 (C2453A or m4, leading to an amino acid change of T461N).
CYP1A1*2A and *2C are in linkage disequilibrium in Caucasians and are associated with an
increased catalytic activity.[7] The variant genotypes have been examined to predict the
expression level of CYP1A1, however CYP1A1 polymorphisms are not good predictors of
CYP1A1 expression.[8] The functional difference between presence of valine or isoleucine in
CYP1A1*2C can be explained by the fact that the valine type of the CYP1A1 enzyme has a
higher catalytic and mutagenic activity towards benzo[a]pyrene than the isoleucine type.
In the past, several review articles have been published about cancer risk in association with
polymorphisms in the CYP1A1 gene, but data are contradictory.[6,9,10] Bartsch et al.[6]
concluded that some CYP1A1/GSTM1*0 genotype combinations seem to predispose the
lung, oesophagus and oral cavity of smokers to an even higher risk of cancer or DNA
damage, requiring, however, confirmation. Houlston et al.[10] perfomed a meta-analysis but
the results of this analysis provide little support for the role of CYP1A1 polymorphisms in lung
cancer risk
In several, more recent studies, an increased risk for lung cancer has been associated with
the CYP1A1*2C allele,[11-17] and *2A.[11,12,16,18] The risk for lung cancer has been more
pronounced in smokers.[11,12,14,18,19] It has been reported that the CYP1A1 genotype and
GSTM1*0 gene-gene interaction result in a greater-than-additive risk for DNA damage and
cancer. An increased phase I enzyme activity of CYP1A1 and a decreased phase II enzyme
activity (GSTM1) could each individually cause an increase in the risk of cancer, but in
combination an even more increased risk for lung cancer has been observed.[14,17,18,20-23] A
study of Taioli et al.[24] found a significant association between CYP1A1*2A and lung cancer
risk in non-smokers. See Table 1. In contrast, no relation between lung cancer risk and
CYP1A1*2A genotype could be detected in a study of Kiyohara et al.[25] and for CYP1A1*2A,
*2C and *4 in a study of Gsur et al.[26], both relatively small studies (N=417 and N=134
respectively).
In the past, several studies have described the eventual association between CYP1A1
genotype and breast cancer risk, but the overall effects are not conclusive.[5,27] Increased
breast cancer risk in women with CYP1A1*2A and the *2C variant genotypes has been
observed in different ethnic subjects (in relation to tobacco smoke exposure)[28-33] and also in
combination with other SNPs in a micro-array based study.[34]
Polychlorinated biphenyls (PCBs) exposure induces CYP1A1 activity, and PCBs themselves
or other xenobiotics are metabolised to carcinogenic intermediates. The majority of studies
have concluded that neither PCBs nor the CYP1A1 genotype alone was independently
associated with breast cancer risk. However, more recent studies reported that, CYP1A1*2C
modifies the association between PCBs exposure and risk of breast cancer in Caucasians.[35-37]
62
63
Exon
Exon 7
SNPs
*2C
A2455G
Nucleotide
I462V
Amino
acid
Caucasian
33 Chl
43.1 T
32.7 T
25.6 Ch
34.2 Ch
Other
Frequency (%)
One mutant allele risk of SCC, also in relation to exposure to tobacco smoking and
GSTM1*0
Frequency in lung cancer patients
risk of SCC in North-Indians, also in combination with bidi-smoking
risk of lung cancer in Chinese women, no interaction with smoking and indoor
airpollution
risk of adenocarcinoma of lung in Turkish people
Frequency in lung cancer patients
risk of lung cancer in Latinos (with inverse relation to smoking exposure) and AfricanAmericans
risk of lung cancer in non-smokers, eventually in combination with GSTM1*0
risk of lung cancer in Chilean people (in combination with GSTM1*0 an even higher risk)
risk of lung cancer in combination with GSTM1*0
Combination with GSTM1*0 risk of females for lung cancer
risk of lung cancer when combined with GSTM1*0
risk of lung cancer and adenocarcinoma in combination with GSTM1*0 and GSTT1*0
risk of breast cancer in women smoking before age of 18
risk of breast cancer in Korean women < 35 years
Homozygous mutants risk of breast cancer in Taiwanese
32% of women had 2 homozygous mutations associated with risk of breast cancer
(COMT, CYP17, CYP19, CYP1A1, and CYP1B1)
Exposure to PCB and mutation risk of breast cancer in postmenopausal women
High PCB serum levels in combination with mutation risk of breast cancer in
postmenopausal women
PCB exposure and mutation risk of breast cancer in Caucasians
Clinical effect
Table 1: Effects of polymorphisms in the CYP1A1 gene
[37]
[35]
[36]
[17]
[18]
[20]
[21]
[22]
[23]
[28]
[30]
[31]
[34]
[16]
[15]
[13]
[12,14]
[11]
Ref
64
Exon
Exon 7
SNPs
*2C
A2455G
Nucleotide
I462V
Amino
acid
Caucasian
20.3 Ind
37.3 Ind
53.4 Tw
79.2 Tw
68.3 Tw
17 Ind
51 Ind
5.5 J
11 J
Other
Frequency (%)
risk of endometrial cancer

risk of prostate cancer
Frequency in prostate cancer patients
risk of prostate cancer in Chilean men (in combination with GSTM1*0)
risk of prostate cancer in Turkish men
risk of prostate cancer in Japanese men and more frequent metastasis
risk of colorectal cancer in Japanese
risk of colon and rectal cancer in currently smoking subjects
risk of oesophageal cancer
risk of oesophageal cancer in combination with GSTM1*0 and heavy smoking
risk of laryngeal cancer in combination with smoking in combination with
GSTM1*0 risk of head and neck cancer
risk for oral cancer
Frequency in oral cancer patients
risk of oral cancer
Frequency in oral SCC
Frequency in oral precancerous lesions
risk of oral SCC in combination with GSTM1*0 and low dose levels of cigarette
smoking
risk of ALL in children
risk of AML in combination with GSTT1*0 and CYP1A1*4
Clinical effect
Table 1: Effects of polymorphisms in the CYP1A1 gene (continued)
[71]
[69]
[65]
[64]
[62]
[63]
[48]
[49]
[50]
[52]
[53]
[59]
[60]
[61]
[44]
[47]
Ref
65
Exon
T3205C
Nucleotide
T461N
Amino
acid
Caucasian
Other
Frequency (%)
risk of breast cancer in African-American women smoking > 20 years

PCB exposure and mutation risk of breast cancer in African-American women
Clinical effect
[29]
[37]
Ref
*4
C2453A
[70]
risk of ALL in girls
[71]
risk of AML (in combination with CYP1A1*2C and GSTT1*0)
9.9
19.1
Frequency in AML patients
T6235C
3-UTR
Higher positivity in lymph node metastasis in mutant allele carriers and
[40]
breast cancer risk in Japanese women
Abbreviations: AML: Acute Myeloid Leukaemia; ALL: Acute Lymphoblastic Leukaemia; Ch: Chinese, Chl: Chilean; Ind: Indian; J: Japanese; PCB: Polychlorinated
biphenyls; SCC: Squamous Cell Carcinoma; Tw: Taiwanese; T: Turkish
*3
SNPs
Table 1: Effects of polymorphisms in the CYP1A1 gene (continued)
Chapter 1.2
Also the CYP1A1*3 genotype was reported to modify effects of PCB exposure among AfricanAmerican women.[37]
In contrast, CYP1A1*2A mutant alleles have been associated with a decreased risk of breast
cancer in Caucasian women[38] and non-whites.[39] In Japanese women a T6235C SNP in the 3flanking region and CYP1A1*2C also showed a reduced risk for breast cancer.[40] No
association between breast cancer and CYP1A1*2A, *2C, *3 and *4 has been detected in a
study of Basham et al.[41], Bailey et al.[42] and in a study of Dialyna et al.[43]
The CYP1A1*2C variant alleles have been shown to predict risk of endometrial cancer.[44]
However, no significant relation between CYP1A1 polymorphisms and gynaecological
malignancies could be detected in other studies.[45,46]
The presence of the CYP1A1*2C mutant allele significantly increased the risk of prostate
cancer.[47-49] The susceptibility to prostate cancer is higher in patients with CYP1A1*2C variant
alleles in combination with GSTM1*0 genotype, compared to individuals carrying CYP1A1 or
GSTM1 alone.[47,48] In a study of Suzuki et al.[50] a significant increased risk of prostate cancer
has been found for both mutated alleles of CYP1A1*2A and *2C. Metastatic prostate cancer
also had a significant association with mutated alleles of CYP1A1*2A and *2C[50] Another
study showed a significant association for CYP1A1 wild-type genotype and an increased risk
for prostate cancer, while the CYP1A1*2A variant allele has been associated with a
decreased risk for prostate cancer in this study.[51]
Sivaraman et al.[52] suggests a potentially important role for CYP1A1*2A and *2C genotype in
the aetiology of colorectal cancer in populations with a high frequency of these
polymorphisms. Based on a mutant CYP1A1 genotype smoking may have a greater impact
on colorectal cancer risk compared to smokers with wild-type CYP1A1.[53] Having wild-type
GSTM1 as well as a CYP1A1 variant allele and the rapid-acetylator NAT2 imputed phenotype,
an increased risk of colon cancer has been found, with the greatest risk being observed in
currently smoking men with a mutant CYP1A1 allele. For rectal cancer an elevated risk has
been observed in men with a variant CYP1A1 and GSTM1 allele and smoking more than 20
pack-years. Contradictory results have been observed in several other studies, where no
significant association has been found for the CYP1A1 genotype and risk for colon or rectal
cancer.[53-56]
Oesophageal cancer has been associated with mutant CYP1A1*2C genotypes, but the level
of statistical significance has not always been reached.[57-59] In the study of Nimura et al.[60] the
combination of CYP1A1*2C and GSTM1*0 lead to a higher incidence of oesophageal
cancer, especially in heavy smokers. Also a higher risk of larynx cancer has been observed
in patients with CYP1A1*2A or *2C alleles.[61] Individuals with the CYP1A1*2C variant allele
were at increased risk for oral cancer and head and neck cancer, especially combined with
GSTM1*0 and GSTT1*0 genotypes.[62-65] However, other studies found no correlation between
the CYP1A1*2C genotype and oral cancer risk,[66] and head and neck cancer risk.[67]
In a study of Infante-Rivard et al.[68] risks for Acute Lymphoblastic Leukaemia (ALL) in
combination with genetic polymorphisms indicated that the effect of parental smoking could
be modified by variant alleles in the CYP1A1 gene, but no statistically significant effect could
66

be observed. In a study of Joseph et al.[69] subjects with CYP1A1*2A and *2C homozygous
variant alleles have been associated with an increased risk of childhood ALL. CYP1A1*2A
genotype in combination with GSTM1*0 alleles is a significant predictor of ALL risk.[70] In a
study of Krajinovic et al.[70] the CYP1A1*4 allele was underrepresented in ALL girls, suggesting
that a gender-specific protective role exists for this allele. In contrast, a higher prevalence of
the CYP1A1*4 allele has been found in AML patients compared to controls. The
combination of CYP1A1*2C, *4 and GSTT1*0 further increased the risk of AML.[71]
In conclusion, the effects of polymorphisms in the CYP1A1 gene on cancer risk are
inconsistent. It seems like the most evidence exists for CYP1A1 polymorphisms to be
associated with lung cancer risk. Many of the published studies have shortcomings, i.e. no
proper documentation (or measurement) of exposure; a mixed ethnic background of the
study subjects; no clear definition of the study population and controls; no adequate sample
size to provide enough statistical power; and not identifying or investigating all
polymorphisms in these genes. These shortcomings have to be avoided in future, in more
defined, larger studies, with the effects of smoking and dietary factors taking into account.
CYP1A2
CYP1A2 activates many dietary and tobacco procarcinogens, notably aromatic and
heterocyclic amines, and nicotine. Exposure to cigarette smoke, polycyclic aromatic
hydrocarbons, pan-fried meat and cruciferous vegetables has been shown to induce CYP1A2
activity in humans.[2]
The CYP1A2 gene is also located on chromosome 15q24 and has 72% sequence identity with
CYP1A1. In the CYP1A2 gene several polymorphisms have been described, of which most
are located in the 5-flanking region: G-3858A (CYP1A2*1C), T-2464delT (CYP1A2*1D), T740G (CYP1A2*1E and *1G), A-164C (CYP1A2*1F). Located in the encoding gene are: C63G
(CYP1A2*2 causing an amino acid change of F21L) and T1545C (CYP1A2*1B, *1G, *1H and
*3 with an amino acid change of D348N). Another SNP found in intron 1 (C-743A), has been
associated with increased catalytic activity towards tobacco smoke.[72] No other associations
between the genotype of CYP1A2 and poor or extensive phenotypes, determined by
measurement of caffeine 3-demethylation, have been observed.[73,74] Because no association
has been identified between differences in CYP1A2 activity and variations in the CYP1A2
structural gene, it has been thought that activities of other carcinogen metabolising enzymes
are involved in the regulation of CYP1A2 activity. MacLeod et al.[75] showed that the GSTM1*0
variant allele or CYP1A1*2C wild-type allele carriers had higher CYP1A2 activity. Upon
exposure to cigarette smoke or high-temperature cooked meat, individuals possessing the
heterozygous CYP1A1*2C variant had a significantly increased CYP1A2 activity.[75]
Patients with C-743A had an increased risk for bladder cancer if they were smokers; if they
also had the slow NAT2 phenotype, the risk increased even more. Besides, when CYP1A2
and NAT2 phenotype were combined, a significantly increased risk on colon cancer has
been found among well-done meat consumers with the rapid-rapid phenotype.[76] However, a
67
Chapter 1.2
lower CYP1A2 activity has been observed in colorectal patients in comparison with
controls.[74]
No associations have been found between CYP1A2 genotype and risk on prostate cancer,[47]
liver cancer,[77] and urothelial cancer.[78]
Gene-gene interactions between GSTM1*0 and CYP1A2 enzyme induction have been
observed in smokers. GSTM1 deficiency not only led to increased hepatic CYP1A2 activity in
current smokers but also to significantly increased levels of bulky polycyclic aromatic
hydrocarbon-DNA adducts in lung parenchyma of smokers and ex-smokers.[6] The gene-gene
interactions mentioned between CYP1A1*2, CYP1A3, GSTM1*0 and NAT are probably
attributable to a greater bioavailability of aromatic inducer compounds, leading to a higher
rate of induction of CYP1A2 in smokers, which in turn increases macromolecular carcinogen
binding.
CYP2C9
The CYP2C subfamily consists of four members in humans: CYP2C8, CYP2C9, CYP2C18 and
CYP2C19. The genes for these proteins are homologues and located on chromosome 10.
The CYP2C9 gene is located at chromosomal region 10q24.2 spanning approximately 55 kb
with 9 exons, and encodes a protein of 490 amino acid residues. CYP2C9 is 92% homologous
to CYP2C19, the expressed product of its neighbouring gene (CYP2C19), differing by only 43
of 490 amino acids. However, the two enzymes have a different substrate specificity.
CYP2C9 appears to play a role, along with other cytochrome P450 enzymes, in the
metabolism of benzo[a]pyrene, a carcinogen in tobacco smoke. The role of CYP2C9 is not
fully defined, as CYP2C9 catalyses detoxification as well as activation steps. Thus, it is not
inconceivable that decreased CYP2C9 activity could increase metabolic activation of
benzo[a]pyrene to carcinogenic intermediates. However, a study of Garcia-Martin et al.[79]
did not identify any association between CYP2C9 polymorphisms and lung cancer risk.
London et al.[80] also showed that the presence of the CYP2C9*2 variant allele, an A144C
amino acid substitution due to a C430T transition in exon 3, is not associated with the risk of
lung cancer; although slight but non-statistically significant elevations in risk were observed
for both African-Americans and Caucasians. To date, the polymorphisms in this gene are not
proven to have any effect on cancer risk.
CYP2C19
The role of polymorphisms in the CYP2C19 gene is questionable. Patients with lung cancer
(squamous cell carcinoma) had a higher frequency of CYP2C19 polymorphisms associated
with poor metaboliser phenotypes, such as the most common defective allele (CYP2C19*2)
a splice mutation in exon 5 (G681A), or CYP2C19*3 a base pair change of G636A in exon 4,
resulting in a stop codon.[81] However, other studies showed no effect on lung and prostate
cancer risk for polymorphisms in this gene, respectively. [82-83]
68
69
Exon
Nucleotide
Caucasian
African
Frequency (%)
Other
Clinical effect
67
87 N
*1B over represented in prostate cancer

risk on prostate cancer
incidence rate of prostate cancer in BPH patients
*1B
5-flanking
A-392G
4
58
Frequency in control patients
4
54
2.9
risk on SCLC in women and men with 20 pack-years
Inversely associated with prostate cancer
5-flanking
C-1232T
5
56
4
56
IVS7 T34G
6
62
5
56
Stop
13
24
G2204C
11
20
Positively associated with prostate cancer
Stop C1454T 0.3
23
0.6
28
C-1232T, *1B, IVS7 T34G,
Haplotype
IVS7 C-202T stop + 766delT
4
13
stop A1639T stop + G2204C 2
7
Abbreviations: BPH: Benign Prostate Hyperplasia; N: Nigerian; SCLC: Small Cell Lung Cancer
SNPs
Table 2: Effects of polymorphisms in the CYP3A4 gene
[87]
[87]
[87]
[87]
[87]
[94]
[85]
[86]
[87]
Ref
Chapter 1.2
CYP3A4
CYP3A4 is involved in the oxidation of testosterone to 2-, 6- and 15-hydroxytestosterone,
which may result in the hormones functional deactivation. Rebbeck et al.[3] hypothesised
that men with the CYP3A4*1B genotype, an A to G point mutation in the 5-flanking region,
may have decreased CYP3A4 protein activity, and thus decreased 2-, 6- and 15testosterone oxidation. This decreased oxidation may in turn increase the bioavailability of
testosterone for conversion to its intracellular mediator, dihydrotestosterone (DHT), the
principle androgenic hormone involved in the regulation of prostate cell growth and
function. This hypothesis is concordant with results that prostate cancer incidence, as well as
allele frequency of CYP3A4*1B, is much higher among Africans than among Caucasians, 3.69.6% in Caucasians, 53-67% in African-Americans and 69-87% in Africans. The allele
frequency of CYP3A4*1B in Asians and Asian-Americans is 0%.[84] The CYP3A4*1B
polymorphism is associated with a higher risk of prostate cancer (Table 2).[85-87] There is no
consensus on direct functional consequences of the CYP3A4 polymorphisms. It has been
reported that the CYP3A4 enzyme is detectable in only 61% of prostate tumours, suggesting
that there is tumour-specific variability in CYP3A4 gene expression.[88] Therefore, response to
hormone therapy may in part be determined by the CYP3A4 genotype or phenotype. The
association of CYP3A4 genotype and prostate cancer may be contributed to a polymorphism
elsewhere in the CYP3A locus, which is linked to the CYP3A4 variant. CYP3A5 is a promising
candidate for this association. Plummer et al.[89] found that the CYP3A4*1B/CYP3A5*3
haplotype was positively associated with prostate cancer, and the CYP3A4*1B/CYP3A5*1
haplotype was inversely associated with risk among Caucasians with less aggressive disease.
Many of the SNPs are in linkage disequilibrium, so some associations may not represent
independent effects, but rather reflect an association with another linked SNP. Another
possible explanation for the lack of association may be polymorphisms in the Pregnane X
Receptor (PXR) gene. PXR regulates the transcription of CYP3A4. Several polymorphisms
have been described that showed varying degrees of reduction in transactivation.[90,91]
Unfortunately, no research has been performed to correlate these polymorphisms with
cancer risk.
No association has been found for the CYP3A4*1B polymorphism and the risk of breast or
ovarian cancer[92] or for hepatocellular carcinoma.[93] In support of this negative finding, in
vitro functional studies indicate that CYP3A4*1B genotype is not a critical factor in the
transcriptional activity of the CYP3A4 5'-flanking region, and is thus unlikely to modulate
CYP3A4-mediated metabolism of steroids.[92]
CYP3A4 also plays an important role in the metabolism of tobacco carcinogens, and
functional CYP3A4 polymorphisms might affect lung cancer risk in individuals.[94] Life-time
tobacco consumption is aetiologically particularly relevant for the histological lung cancer
types squamous cell carcinomas and small cell lung cancers. For women, an increased lung
cancer risk independent of smoke status has been revealed in CYP3A4*1B carriers.[94] Heavier
smoking men with the CYP3A4*1B allele had a significantly higher risk of lung cancer
compared to wild type carriers with lower tobacco exposure (<20 pack-years).
70

To date, although different effects of CYP3A4 polymorphisms have been described, it is not
clear that these polymorphisms explain interindividual differences in the development of
cancer.
CYP3A5
CYP3A5 represents at least 50% of the total hepatic CYP3A content in humans. The CYP3A5
protein is polymorphically expressed in adults with readily detectable expression in about
10-20% in Caucasians, 33% in Japanese and 55% in African-Americans. The primary causal
mutation (CYP3A5*3) confers low CYP3A5 protein expression as a result of improper mRNA
splicing and reduced translation of a functional protein.
Lung cancer patients had a significantly lower frequency of CYP3A5*1 expression than
healthy controls.[95] In contrast, Dally et al.[94] found a non-significantly increased lung cancer
risk for homozygous CYP3A5*1 allele carriers. Also no statistically significant difference
between healthy controls and other cancers (AML, CML,[96] hepatoma, cervical, breast, oral
and thyroid[95]) has been observed.
Overall, no clear associations have been detected between polymorphisms in the CYP3A5
gene and cancer risk. Linkage disequilibrium with CYP3A4 variants may elucidate more
polymorphisms associated with cancer risk.
Phase II enzymes
Uridine diphosphate glucuronosyltransferase (UGT)
UGT1A1
UGT1A1 is involved in the glucuronidation of bilirubin and in the inactivation of oestradiol
(E2) and its oxidised metabolites. Inherited alterations in the expression of UGT1A1 result in
interindividual differences in oestrogen metabolism that ultimately may result in
interindividual differences in endometrial cancer risk. The UGT1A1*28 allele (a (TA)7
tandem repeat instead of (TA)6 in the promoter region) has been inversely associated with
endometrial cancer risk, indicating that the decreased expression of the UGT1A1 protein can
potentially have a beneficial effect on the uterus.[97] The association between the UGT1A1
genotype and endometrial cancer was stronger among postmenopausal women compared
with premenopausal women. See table 3.
Oestrogen is also an important factor in the aetiology of breast cancer. By metabolising
oestrogen, UGT1A1 represents a candidate gene in breast carcinogenesis. The (TA)7 allele
has indeed been associated with an increased risk of developing breast cancer in Chinese
and African premenopausal women.[98,99] Among African premenopausal women, this
association was stronger for oestrogen receptor negative (ER-) breast cancer than ER+ breast
cancer.[98] In the study of Adegoke et al.[99] the elevated risk was primarily seen in Chinese
71
72
9.0
8.0
(TA)6TAA
(TA)5TAA
(TA)6TAA
(TA)8TAA
Promoter
Promoter
*33
*34
Abbreviations: Ch=Chinese
42
38
(TA)6TAA
(TA)7TAA
Promoter
3.0
2.0
African
*28
32-34
27
Caucasian
Frequency (%)
Nucleotide
Exon
SNPs
13.0 Ch
12.5 Ch
Other
Table 3: Clinical effects of SNPs in the UGT1A1 gene
[98]
[98]
2-fold risk on breast cancer with mutant allele in AfricanAmerican women

Frequency in breast cancer patients
[99]
[100]
[99]
[98]
[97]
Ref

Risk of endometrial cancer 6/6 > 6/7 > 7/7

Frequency in control patient groups
Frequency in endometrial cancer patient group
2-fold risk on breast cancer with mutant allele in AfricanAmerican women.
risk on breast cancer among women < 40 with UGT1A1*28 allele
risk of ER- tumour in postmenopausal women
Clinical effect
Pharmacogenetic screening in cancer treatment and risk

women who had a later menarche, short menstrual years, absence of family history of breast
cancer, low waist-to-hip ratio, or low body-mass index. Also patients with UGT1A1*33 (a
(TA)5 repeat) and *34 (a (TA)8 repeat) mutant alleles have an increased risk on breast
cancer.[98] It has been suggested that lower activity alleles can lead to higher amounts of
oestradiol in the breast tissue, thereby potentially altering the micro-environment, and
increasing the risk of malignancy. In contrary to these results, a study of Sparks et al.[100]
showed that the UGT1A1*28 allele reduced the risk of ER- breast cancer in postmenopausal
women of different ethnicities. Besides, a more recent study of Guillemette et al.[101] showed
no significant association between the UGT1A1*28 mutant allele and breast cancer in
Caucasian women.
The effect of the UGT1A1 genotype on breast cancer risk is different per ethnicity. Further
research has to be performed in order to clarify the influence of ethnicity and the UGT1A1
genotype on oestradiol metabolism and endometrial and breast cancer risk.
Glutathione S-Transferase (GST)

In humans, six classes of soluble GSTs (Alpha (A), Mu (M), Pi (P), Omega, Theta (T) and
Zeta) are known, with compelling evidence of functional polymorphic variation.[102] Many
potential human carcinogens are detoxified by GSTs, and deficiency of GST enzymes may
therefore affect the metabolic fates of these chemicals and raise cancer risk in exposed
individuals. See table 4 for the effects of polymorphisms on cancer risk.
GSTM1
Lack of GSTM1 activity, caused by an inherited homozygous deletion of the GSTM1 gene
(null genotype or *0), has been associated with both an increased as well as a decreased
risk of several forms of cancer. Decreased risk has been associated with isothiocyanates
(ITCs), which are a derivate of cruciferous vegetables that increase the expression of GSTM1,
and are known to have cancer-protective effects.[103]
The *0 genotype of GSTM1 was found to be a significant predictor of ALL and Chronic
Lymphocytic Leukaemia (CLL) risk.[69,104,105] GSTM1*0 in combination with CYP1A1*2A is also
a predictor of ALL risk.[70] In contrast, in another study a reduced risk was observed for ALL,
glial brain tumours and osteosarcoma in patients carrying the *0 allele of GSTM1.[106]
The risk of developing lung cancer in correlation with the GSTM1 genotype has been
thoroughly investigated,[107] and a higher risk has been observed in patients with the
GSTM1*0 genotype.[18,19,25,108,109] In a study of Spitz et al.,[110] a low intake of ITCs, combined with
the GSTM1*0 genotype has been associated with increased lung cancer in current smokers.
A higher weekly intake of ITCs reduced the risk of lung cancer to a greater extent in smokers
than non-smokers.[111] A gene-environment interaction has been suggested, with combined
GSTM1*0 genotype and high-dose environmental tobacco smoke ( 40 pack-years by
husbands) conferring significantly higher risk compared to GSTM1 non-null genotype and
low-dose tobacco smoke exposure.[25] GSTM1*0 in combination with CYP1A1*2C showed an
elevated risk of lung cancer.[17,18,20,22] The combination of GSTM1*0, CYP1A1*2A and GSTT1*0
73
74
M1
SNPs
Nucleotide
Amino
acid
[69]
[104]
[105]
[70]
[106]
[18]
Null genotype risk of childhood ALL

Null genotype risk of developing CLL
Null genotype risk of ALL (in combination with CYP1A1*2C even )
risk of ALL, glial brain tumours and osteosarcoma for null allele
Null genotype in combination with CYP1A1*2A or *2C risk of lung cancer, in relation to exposure of
tobacco smoke
Null genotype risk of SCC in light smokers
Null genotype risk of lung cancer in relation to smoking
2.6-fold risk of laryngeal cancer with null genotype
risk for lung adenocarcinoma for null genotype
Low ITC intake with GSTM1 null genotype: lung cancer risk in current smokers
High ITC intake with GSTM1 null genotype: risk of lung cancer in Chinese females
Combination of null genotype and CYP1A1*2C risk of lung cancer
Combination of null genotype and CYP1A1*2C risk of smoking-related lung cancer
Null genotype (in combination with CYP1A1*2C) risk of lung cancer in Chinese
risk of adenocarcinoma of lung (in combination with GSTT1*0 and CYP1A1*2A)
risk of SCC with null genotype, stronger with UV light exposure and in smokers
risk of breast cancer in postmenopausal women with null genotype in combination with GSTP1*A
and GSTT1*0
Null genotype in combination with GSTT1*0 risk of cervical carcinoma in premenopausal women
Null genotype risk of bladder cancer
Null genotype risk of prostate cancer in combination with CYP1A1*2C in Japanese
Null genotype risk of prostate cancer in Chinese (in combination with CYP1A1*2A even )
Null genotype: 36% in prostate cancer risk
[45]
[114]
[47]
[48]
[115]
[19]
[25]
[108]
[109]
[110]
[111]
[17]
[20]
[22]
[23]
[112]
[113]
Ref
Clinical effect
Table 4: Clinical effects of SNPs in the GST genes
75
I105V
I105V
V113A
A313G
C341T
P1*C
Amino
acid
A313G
Nucleotide
P1*B
M1
SNPs
[63]
[66]
Null genotype risk of oral cancer in combination with CYP1A1*2C in Indian subjects
Null genotype in combination with smoking risk of oral cancer
Null genotype in combination with CYP1A1*2C and heavy smoking risk of oesophageal
carcinoma
Null genotype in combination with CYP1A1*2C risk of head and neck SSC
Non-null genotype in combination with CYP1A1*2C and NAT2 rapid-acetylator risk of colorectal
cancer
*B risk of childhood ALL
Wild-type risk of developing CLL
2.8-fold risk of laryngeal cancer with homozygous V105 genotype
GSTP1*B allele strongly associated with bladder and testicular cancer
Frequency of wild-type lower than of GSTP1*B polymorphism in squamous cell cancer of the
oral/pharynx and larynx
GSTP1*A homozygotes develop larger number of SCC
of breast cancer in women used HRT and genotype COMT-L and GTSP1*A or GSTT1 null
genotype
*C under-represented in ALL patients
risk of development of squamous cell carcinoma
[121]
[118]
[112]
[124]
[121]
[105]
[108]
[122]
[123]
[60]
[62]
[53]
Ref
Clinical effect
76
Nucleotide
Amino
acid
[125]
[19]
[23]
Null genotype over-represented in lung and larynx cancer

Null genotype risk of lung cancer in light Caucasian smokers, in heavy smoker risk of lung cancer
Null genotype risk of lung cancer in advanced age of diagnosis and in combination with CYP1A1*2A
and GSTM1*0
risk of ALL, glial brain tumours and osteosarcoma for non-null allele
Null genotype risk of AML in combination with CYP1A1*2C and *4
Null genotype risk of developing CLL
Null genotype in combination with heavy smoking pancreatic cancer (women>men)
Lower GSTT1*0 frequency in oesophageal adenocarcinoma
Null genotype in combination with CYP1A1*2C and GSTM1*0 risk of oral cancer
Null genotype risk of bladder cancer
GSTT1 null genotype risk of lung cancer
Null genotype risk of colon cancer
HPV positive women with GSTM1*0 and GSTT1*0 risk developing cervical cancer before age 40
years
GSTT1 null genotype in combination with GSTM3*B and GSTP1*A risk of breast cancer in
premenopausal women
of breast cancer in women used HRT and genotype COMT-L and GTSP1*A or GSTT1 null genotype
[124]
[113]
[69]
[106]
[71]
[105]
[126]
[57]
[63]
[114]
[110]
[55]
[45]
Ref
Clinical effect
Abbreviations: AML: Acute Myeloid Leukaemia; ALL: Acute Lymphoblastic Leukaemia; CLL: Chronic Lymphocytic Leukaemia; HPV: Human
Papilloma Virus; HRT=Hormone Replacement Therapy, ITC: isothiocyanates; SCC=Squamous Cell Carcinoma, UV=Ultra Violet
T1
SNPs

in heavy smokers is involved in an increased development of lung cancer.[23] Studies of Yang
et al.[13] and Gsur et al.[26] showed no association between the GSTM1 genotype, smoking
status and lung cancer risk.
The GSTM1 null genotype has also been associated with increased squamous cell carcinoma
(SCC) risk.[57,112] This association was particularly strong in patients with higher ultraviolet
light exposure and in smokers. The genotype and sunbathing score were synergistic and
immunosuppressed individuals with both risk parameters demonstrated an even higher risk
of development of SCC.[112]
Mitrunen et al.[113] found a significant correlation between the GSTM1 null genotype and a
higher risk of breast cancer in postmenopausal women. Also a higher risk for cervical
carcinoma has been found in premenopausal women with the GSTM1 genotype.[45] Another
study showed an individual susceptibility to bladder cancer modulated by GSTM1*0
genotypes.[114] The susceptibility to prostate cancer is higher in patients with the GSTM1*0
genotype in combination with CYP1A1*2C variant alleles.[47,48] A 36% reduction in prostate
cancer risk has been associated with the GSTM1*0 genotype in Finnish male smokers.[115]
For oral cancer risk an increased association has been found for GSTM1*0 in combination
with cigarette use,[63,66] and for oesophageal, head and neck cancer in combination with the
CYP1A1*2C genotype.[60,62]
GSTM1 present in combination with the CYP1A1*2C variant allele and the rapid-acetylator
NAT2 phenotype, caused an elevated risk of colon cancer and rectal cancer among
smokers.[53]
Besides all the studies showing a correlation between GSTM1 null genotype and cancer a
lack of association has been found between the GSTM1 null genotype and risk of prostate
cancer,[49] colorectal cancer,[56] (advanced) breast cancer,[43,116,117] basal cell carcinomas,[118] oral
cancer,[119] head and neck cancer,[67] and bladder cancer.[120] Inconsistencies in these studies
about the role of GST genotypes in cancer could be due to unexpected confounding from
dietary factors or smoking. Besides, a limited sample size can also be responsible for
suggestive findings. A larger sample size, with ethnicity as covariate, would be required to
corroborate the non-significant results. Overall, GSTM1*0 is a moderately strong
susceptibility factor, but may become a dominant risk factor in the presence of certain genegene combinations, which results in increased DNA damage and mutational events in target
and surrogate tissues.
GSTP1
The polymorphism GSTP1*B differs from GSTP1*A (wild-type) by a single A to G transition at
nucleotide +313 (I105V). GSTP1*C has transitions A313G and C341T, respectively leading to
amino acid changes I105V and V113A.
The genetic variants of GSTP1, which are expressed at the protein level, appear to contribute
differently to the risk of ALL, probably because of distinct substrates specificities. The
GSTP1*B genotype has been found to be a predictor of ALL risk, and the GSTP1*C genotype
77
Chapter 1.2
has been underrepresented in ALL patients in the same study.[121] The combination of
GSTP1*B and GSTM1 null genotypes further increased the risk of ALL. Also the risk of
Chronic Lymphocytic Leukaemia (CLL) increased with the putative GSTP1 genotype.[105] An
increased frequency of GSTP1*B homozygous subjects has also been observed among
patients suffering from lung cancer, oral/pharyngeal and laryngeal carcinomas, bladder
cancer, testicular cancer, and some other diseases related to cigarette smoking.[108,122,123]
Ultraviolet radiation induces free radical damage, which is a major mechanism of skin
carcinogenesis. The genetically determined ability to metabolise free radicals may also
predispose to skin cancer. The GST enzymes play a major role in limiting the toxic effects of
reactive oxygen species. For example, the GSTP1*C allele has been associated with the
development of SCC.[118] Homozygous GSTP1*A carriers also developed larger numbers of
SCC with lower ultraviolet light exposure and cigarette consumption.[112]
In premenopausal women the risk of breast cancer rose with the combination of the
genotypes GSTM3*B, GSTP1*A and GSTT1 null. A substantially increased risk of breast
cancer was also seen for women who had used hormone replacement therapy (HRT) and
had the GSTP1*A genotype or the GSTT1 null genotype.[124] These associations appeared to
be mainly attributable to long-term users of HRT.
No significant associations were found between the GSTP1*B polymorphism and prostate
cancer risk.[115]
In conclusion, GSTP1 polymorphisms has been associated with an increased risk of several
forms of cancer, such as SCC and ALL, and no associations has been found for prostate
cancer. This can possibly be explained by the fact that the variants are supposed to decrease
GSTP1 activity for some substrates, but not for others. Thus, their actually importance is not
fully understood. Combinations of polymorphisms can possibly further increase the cancer
risk, hence screening for these polymorphisms is necessary to explore the complete effects
on risk for cancer.
GSTT1
Homozygous deletion of the GSTT1 gene (null genotype or *0) was found to be overrepresented in lung and larynx cancer.[125] In a study of Alexandrie et al.[19] the GSTT1*0
genotype appeared to be a possible risk factor for lung cancer in light smokers, whereas, in
heavy smokers, this genotype has been associated with decreased risk for lung cancer.
Different effects have been seen when GSTT1*0 is combined with CYP1A1*2A and GSTM1*0,
the variant alleles showed an elevated risk in development of lung cancer.[23]
An increased risk of ALL, glial brain tumours and osteosarcoma for patients carrying the nonnull allele of GSTT1 has been observed.[69,106] The null genotype of GSTT1 has been more
frequently observed in AML patients than in controls. The combination of GSTT1*0 and
CYP1A1 mutant alleles further increased the risk of AML in adults.[71] In a study of Yuille et
al.[105] it was found that carrying more than one of the putative high-risk GST genotypes
(GSTM1, GSTT1 and GSTP1) significantly increased the risk of developing CLL, the risk being
highest in persons possessing all 3 high-risk genotypes. An interaction between pancreatic
78

cancer and GSTT1*0 genotype with cigarette smoking among Caucasians has been observed
that was more prominent among women than among men.[126] Susceptibility to oesophageal
adenocarcinoma,[57] oral cancer,[63] bladder cancer,[114] lung cancer,[110] colon cancer[55] cervical
cancer,[45] and breast cancer[113,124] was also modulated by the GSTT1*0 genotype.
No significant associations were found between the GSTT1*0 polymorphism and breast
cancer,[43] oral cancer,[66] head and neck cancer,[67] SCC,[118] or prostate cancer risk.[115] In
general, these studies were performed in a smaller population compared with studies that
showed increased risk of cancer for GSTT1*0. Consequently, significant differences in cancer
risk in the smaller population studies could probably not be detected.
Overall, several cancer risks has been associated with GSTT1*0, but others did not have
associations with this polymorphism. This may be caused by the experimental design of the
different studies. Often larger studies are needed to obtain more precise risk estimates.
Besides, it is more likely, however, that the majority in variability in susceptibility to cancer is
due to (polymorphisms) in genes that have yet to be identified or tested.
Sulphotransferase (SULT)
Sulphation plays an important role in the metabolism and bioactivation of many dietary and
environmental mutagens, including heterocyclic amines, which are implicated in the
pathogenesis of colorectal and other cancers.
SULT1A1
Epidemiological studies investigating a possible association of SULT1A1 polymorphisms and
malignancy have not been conclusive. See table 5.
A G638G SNP (SULT1A1*2) in the SULT1A1 gene causes a R213H amino acid change and
encodes for an allozyme with low enzyme activity and stability compared to the wild-type
enzyme. Therefore this SULT1A1 genotype can influence susceptibility to mutagenicity
following exposure to heterocyclic amines, polycyclic hydrocarbons and arylamines from
cigarette smoke and other environmental toxins.
A significantly reduced risk of colorectal cancer has been associated with homozygosity for
SULT1A1*1 (wild-type) in subjects under an age of 80 years.[127]
Large-scale studies have investigated the effect of the SULT1A1*2 polymorphism on breast
cancer risk in women. Although Zheng et al.[128] and Tang et al.[129] found that this
polymorphism is associated with increased breast cancer in Caucasian women, Seth et al.[130]
did not. A study in Chinese subjects observed a positive association between the mutant
allele and the risk of breast cancer in Chinese women.[131] This effect of a high risk of breast
cancer with a mutant allele might be modified by the exposure level of endogenous
oestrogens and heterocyclic amines in these studies.[128] The effect of smoking and the mutant
SULT1A1 allele in breast cancer has been associated with low sulphation activity and a 2-fold
excess risk of breast cancer compared to never smokers carrying the wild-type genotype. The
mutant allele increased the risk only in premenopausal women independent of smoking
79
80
Exon
SNPs
*2
G638A
Nucleotide
R213H
Amino
acid
[127]
[128]
[129]
[131]
[132]
[133]
[134]
[135]
risk of colorectal cancer in patents < 80 years

Homozygosity risk factor for breast cancer
number of variant alleles associated with breast cancer risk
risk of breast cancer in Chinese women
Risk on breast cancer in smoking premenopausal women with mutant allele
lung cancer dependent on cumulative smoking dose
lung cancer risk for mutant allele
risk developing oesophageal cancer in Taiwanese, in non-smokers, non-drinkers and nonchewers
Mutant allele risk of bladder cancer in women
Mutant allele bladder cancer risk in never smokers in women
risk bladder cancer in never smokers
[114]
[136]
[137]
Ref
Clinical effect
Table 5: Clinical effects of the SNPs in the SULT1A1 gene
Pharmacogenetic screening in cancer treatment and risk

status.[132] On the contrary, a non-statistically significant risk reduction on breast cancer with
the variant SULT1A1*2 allele has been observed in a study of Sparks et al.[100]
The variant allele has also been associated with a significantly increased risk for overall lung
cancer. The risk was more pronounced in younger subjects and limited to smokers.[133]
Furthermore, the risk of lung cancer was increased consistently with cumulative smoking
dose.[133] These results are consistent with earlier published data from Wang et al.[134]
Wu et al.[135] reported that polymorphisms in the SULT1A1 gene influence the risk of
developing oesophageal cancer in Taiwanese males. This positive association was found to
be even stronger among non-smokers, non-drinkers or non-chewers (areca).
The mutant SULT1A1*2 allele showed a marginal protective effect in bladder cancer risk.[114]
However, in a study of Zheng et al.[136] a statistically significant reduced risk of bladder cancer
was observed only in women and not in men with the mutant allele. In addition, there was a
reduced bladder cancer risk in never smokers with the mutant allele, but not in former or
current smokers.[137] The mutant allele also appeared to provide some protective benefit for
current and former smokers, as compared to those with the wild-type genotype, but this was
not statistically significant.
No statistically significant effect has been found for hepatic, colon, lung, oral, gastric, renal,
urothelial, prostate and cervical cancerous patients with SULT1A1*2 (and SULT1A2*2).[137-140]
The association between polymorphisms in SULT and neoplasias appears to be complex and
varies between subgroups. This is not surprising, as SULTs are involved in the activation of
some carcinogens, in the inactivation of other carcinogens, and the regulation of many
hormones.[141] Further research is necessary to explore the effects of polymorphisms on the
risk of cancer.
Arylamine N-Acetyltransferase (NAT)

Two NAT iso-enzymes have been identified in humans, namely NAT1 and NAT2, which are
products of distinct genetic loci, designated NAT1 and NAT2, respectively. The two
functional NAT genes share 87% nucleotide identity, which translates to an 81% homology at
the amino acid level. Both enzymes catalyze N-acetylation (usually deactivation) and Oacetylation (usually activation) of aromatic and heterocyclic amine carcinogens.
The association between acetylator status and the risk of various diseases has been
extensively reported. Altered risk with either the slow or rapid metaboliser phenotype has
been observed for bladder, colon and breast cancer, but also for other diseases like systemic
lupus erythemaetosis, diabetes, Gilberts syndrome, Parkinsons disease and Alzheimers
disease.[142] The associations with these disorders imply a role for environmental factors that
are metabolised by the NATs, in particular NAT2. However, identifying those factors remains
elusive. Humans are exposed to many toxic NAT substrates, including food-derived
heterocyclics present as well as arylamines such as 4-aminobiphenyl and -naphthylamine
81
Chapter 1.2
present in tobacco smoke. Moreover, occupational exposure to arylamine carcinogens such
as benzidine has also been reported.[142]
NAT1
To date, 26 different NAT1 alleles have been detected in human populations
(www.louisville.edu/medschool/pharmacology/NAT.html), however, only a small number
have been shown to alter phenotype in vivo. A population study showed a distribution of
NAT1 activity that was clearly bimodal in nature, with 8% of the individuals being slow
acetylators.[143] Immunochemical detection of NAT1, but not of NAT2, has been reported in
human urinary bladder.[144] It has been suggested that the local NAT1 activity in the bladder
contributes to the formation of highly reactive acetoxy esters and that polymorphic NAT1
genotypes could therefore modulate an individuals risk to develop bladder cancer. This is
confirmed by the fact that the NAT1*10 genotype, associated with a nucleotide change of
T1088A and C1095A and an increased activity of NAT1, was over-represented among bladder
cancer patients.[145,146] Also an increased risk on urinary bladder cancer risk combined with
smoking exposure has been found in Japanese patients with a NAT1*10 allele.[146,147] However,
for a Caucasian population results have been conflicting, NAT1*10 has not been associated
with bladder cancer risk,[148,149] but a study of Cascorbi et al.[149] reported that individuals with
NAT1*10 are at a significantly lower risk for bladder cancer.
The relationship between NAT1 polymorphism and breast cancer risk has also been
explored. See table 6. Millikan et al.[150] observed no relationship of smoking, NAT1 genotype
and a higher risk for breast cancer, except among postmenopausal women who were
smoking in the past 3 years with the NAT1*10 allele. The NAT1*10 has also been associated
with an elevated risk of breast cancer among former or light smokers.[151] In a study of Lee et
al.,[152] the rare NAT1*11 allele (a combination of the following polymorphisms: C344T, A-40T,
G445A, G549A, T640G, 9(1065-1090) and C1095A with normal enzyme activity) has been
observed three times more frequent in breast cancer patients than in controls. Also an
elevated risk of breast cancer has been observed in postmenopausal women with the
NAT1*11 allele who smoke or who consumed a high level of well-done meat.[151]
Bell et al.[153] found an association between the NAT1*10 allele and colorectal cancer.
Another study showed a higher risk for colorectal cancer in individuals who consumed welldone meat and possessed both the NAT1*10 allele and the rapid NAT2 acetylator
phenotype.[154] NAT1*10 and NAT2*4 (the polymorphisms for the rapid NAT2 acetylator) are
in linkage disequilibrium,[155,156] which may be an important factor in the association of the
NAT1*10 allele with colorectal cancer. The disparity in results may relate to misclassification
of NAT1*10 alleles, because the most commonly used test to detect the NAT1*10 allele does
not distinguish between the rapid acetylator NAT1*10 and the slow acetylator NAT1*14A
(both containing the polymorphisms T1088A and C1095A, with *14A also containing
G560A).[143]
No association has been observed between NAT1*10 genotype and risk for bladder,[148]
colon[157,158] and larynx cancer.[155]
82
83
*14
*15
C-344T, A-40T, 445A,

G459A, T640G, 9
(1065-1090), 1095A
G560A
C559T
R187Q
R187Q
V149I
S214A
None
T1088A
C1095A
*10
*11
Amino acid
Nucleotide
SNPs
Ref
[145]
[146]
[147]
[149]
[150]
[151]
[153]
[154]
[160]
[151]
[152]
[159]
[159]
Clinical effect
In bladder cancer patients mutant allele is over represented
risk on bladder cancer in patients with mutant allele (and smoking)
risk on urinary cancer among smokers
risk for bladder cancer, particularly when exposed to environmental risk factors
risk on breast cancer in postmenopausal women smoking in the past 3 years and with
*10 allele
risk on breast cancer among former or light smokers
risk on colorectal cancer in combination with NAT2 rapid acetylator
risk on colorectal cancer with NAT2 rapid acety;ator and well-done meat consumption
risk on prostate cancer for rapid acetylator
risk on breast cancer in postmenopausal women who smoke or eat well-done meat
Mutant allele 3-times more frequent in breast cancer patients
risk on lung cancer in slow acetylators
risk on lung cancer in slow acetylators
Table 6: Effects of the SNPs in the NAT1 gene
Chapter 1.2
A correlation between slow acetylator NAT1 genotype and an increased risk of developing
cancer has also been observed for lung cancer,[159] in contrast, for prostate cancer an
increased risk has been found for the rapid acetylators.[160] Although polymorphisms in the
NAT1 gene have been described, it is not clear that these polymorphisms explain
interindividual differences in development of cancer.
NAT2
To date, 29 different NAT2 alleles have been detected in human populations. Each of the
variant alleles is comprised of between one and four nucleotide substitutions, of which 13
have been identified within the 870 bp coding region of the gene. The following
polymorphisms, that lead to a change in the encoded amino acid have been identified as
slow acetylators: *5 (T341C resulting in an amino acid change of I114T), *6 (G590A causing
an amino acid change of R197Q),*7 (G857A resulting in an amino acid change of L268R),
*10 (G499A leading to an amino acid change of E167L), *14 (G191A and A803G leading to a
change of respectively R64Q and I114T), *17 (A434C with a change of Q145P), *18 (A845C
causing an amino acid change of L282T) and *19 (C190T causing a R64W change). The
majority of studies investigating the relationship between NAT2 genotype and disease risk
use PCR-based assays that detect only three SNPs (C481T, G590A and G857A) to infer NAT2
acetylation status. When none of these SNPs are present, wild-type NAT2*4, a high-activity
(rapid) allele, is designated.[161] Although several NAT2 SNPs are in linkage disequilibrium,
assessment of only these three SNPs results in misclassification of different NAT2 alleles.
Deitz et al.[162] performed an 11 SNP assay (instead of only 3 SNPs) and showed that a
substantial decrease in sample size (minus 22%), needed to detect a previously reported
NAT2 smoking interaction for bladder cancer, could be obtained.
Most publications mention the phenotype, slow or rapid acetylator, and not the genotype.
Several studies have investigated a possible association between NAT2 acetylator phenotype
or genotype and cancer, but the findings have been very inconsistent.
An association between slow acetylator phenotype and urinary bladder cancer has been
observed and is the strongest in studies in which there are documented exposures to
aromatic amine carcinogens. The possible mechanism for this association is that slow NAT2
acetylators of aromatic amine carcinogens compete poorly with metabolic activation via
CYP and/or prostaglandin H-synthases. NAT2 genotype studies [148,163-165] also show associations
consistent with earlier phenotypic studies, in which urinary bladder cancer risk are highest
for particular NAT2 alleles associated with slow acetylator phenotype(s).[166] A meta-analysis
of several studies showed a slight, but statistically significant, over-representation of NAT2
slow acetylators in bladder cancer patients.[167] Cascorbi et al.[149] also showed that individuals
wild-type for NAT2 (NAT2*4) were at a significantly lower risk for bladder cancer.
Several studies found an association between rapid NAT2 acetylator phenotype and
colorectal cancer, whether in combination with consuming well-done meat or not.[55,76,154,168-174]
A study of Gil et al.[173] found that the association with colorectal cancer was limited to
homozygous rapid (NAT2*4/*4) acetylators.
84

Rapid acetylator phenotype has also been associated with breast cancer risk.[175,176] Recently,
the association between NAT2 acetylator genotype and breast cancer has been investigated
in relation to smoking and diet.[177] NAT2 genotype alone has not been associated with
increased breast cancer risk,[178] whereas the risk was increased for current and ex-smoking
subjects with a slow acetylator phenotype and in contrast, passive smoking showed an
increase in breast cancer risk in rapid acetylators. The relationship between NAT2 genotype
and breast cancer among smoking women shows conflicting results: two studies reported a
higher risk among slow acetylators,[179,180] and one reported a higher risk among rapid
acetylators.[150] The rapid/intermediate NAT2 genotypes also have been associated with
increased breast cancer risk in women who consistently consume very well-done meat.
Results of Lee et al.[152] suggest that genetic polymorphisms of NAT1 and NAT2 have no
independent effect on breast cancer risk, but that they modulate breast cancer risk in the
presence of GSTM1 and GSTT1 null genotypes.
The role of NAT2 phenotype in susceptibility to lung cancer showed a slight over
representation of rapid acetylators. A subsequent NAT2 genotype study of Cascorbi et al.[181]
showed clearly that the highest risk was found in smokers with the homozygous rapid
acetylator (NAT2*4/*4) genotype. Similarly, Nyberg et al.[182] and Zhou et al.[183] reported an
increased risk for lung cancer for rapid acetylators who were smokers. However, according
to Zhou et al.,[183] the rapid acetylator genotype is protective against lung cancer in nonsmokers. Combinations of NAT2 slow acetylation and SULT fast sulphation (*1/*1) with
smoking were shown to result in a 4 times higher risk of adenomas compared to never
smokers with other inherited gene variants, although this effect was not statistical
significant.[184]
Head and neck cancers are strongly associated with smoking, and several studies have
explored the role of NAT1 and NAT2 polymorphisms in the incidence of head and neck
cancer in smokers. The slow NAT2 acetylator phenotype has been associated with the
development of head and neck cancer in Caucasians[185-187] and with the development of
oesophageal[188] and laryngeal[189] cancers in Japanese. However, the homozygous rapid
acetylator genotype (NAT2*4/*4) was strongly associated with laryngeal cancer in two
studies.[155,156]
No relationship between NAT2 genotype and colorectal,[190-195] breast[196-198] (independent on
meat consumption[152,199,200] or smoking),[201] lung,[159,202] oral cavity,[203] and prostate cancer[83,204]
have been observed.
Overall, no conclusive effects have been described for the association of NAT2 genotypes
and cancer risk. Further research is necessary to explore the effects of polymorphisms on the
risk of cancer, in combination with exposure to for example smoking and well-done meat
consumption.
85
Chapter 1.2
Drug transporters
P-glycoprotein (P-gp, MDR1)
P-glycoprotein (ABCB1) is a 1280 amino acid, glycosylated plasma membrane protein,
which consists of two similar halves, each containing six putative transmembrane regions
and an ATP binding/utilization domain, separated by a flexible linker polypeptide.[205] ATP
binding and hydrolysis appear to be essential for the proper functioning of P-gp, including
drug transport. P-gp is a member of the ATP binding cassette (ABC) family of transporters.
Substrates for P-gp cover a variety of chemical structures, and have diverse therapeutic
indications. The molecules are usually lipophilic and amphipathic, containing one or more
aromatic rings. Among the vital organs expressing P-gp are the intestinal epithelium of the
stomach, small intestine and colon, the liver bile canalicular membrane, the blood brain
barrier (BBB) and the kidney.[206-208] P-gp has been implicated in multidrug resistance (MDR)
by transporting xenobiotics out of the cells.
A number of studies have indicated that overexpression of P-gp caused by MDR1 gene
amplification can be applied as a prognostic marker in certain diseases, such as leukaemia
or ovarian cancer.[209]
The silent C3435T polymorphism in exon 26 (*6) has been reported to be a risk factor for
cancer.[4] In a study of Jamroziak et al.[210] the mutant homozygous TT genotype of the *6
polymorphism has been found to be associated with occurrence of ALL
The mutant *6 allele has been associated with a risk for non-Clear Cell Renal Cell Carcinoma
(CCRCC), with the highest risk for homozygous mutant allele carriers.[211] In patients with
colorectal carcinoma no difference in C3435T polymorphisms have been found between
normal tissues and tumour tissue samples.[212]
Overall, polymorphisms in the P-gp gene have been associated with cancer risk, such as ALL
and CCRCC. The research for consequences of polymorphisms on cancer risk has to be
expanded. Screening of polymorphisms has to be performed not only for the MDR1 gene,
but also in other drug metabolising and drug transporter genes. It may be important to link
polymorphisms and thereby clarify the role of the different polymorphisms on cancer risk.
Obviously, more cancer types need to be investigated for the role of polymorphisms in the
MDR1 gene on cancer risk.
Breast Cancer Resistance Protein (BCRP; ABCG2)

The multidrug transporter BCRP (encoded by ABCG2) is responsible for the active secretion
of clinically and toxicologically important substrates. 2-amino-1-methyl-6-phenylimidazol[4,5b]pyridine (PhIP) is abundantly present in cigarette smoke and well-done meat and is
mutagenic and carcinogenic in rodents, inducing lymphomas in mice and colon, mammary
and prostate carcinomas in rats. PhIP has also been implicated in human breast
carcinogenesis. In mice, BCRP effectively restricts the exposure to ingested PhIP.[213] BCRP is
also strongly induced in the mammary gland of humans during lactation and is actively
86

concentrating PhIP into milk.[214] Genetic variations in the ABCG2 gene can cause a
decreased function of the BCRP enzyme and thereby improving food safety for breast-fed
infants.
Intra- or interindividual differences in BCRP activity due to genetic variations may thus also
affect the exposure to PhIP and related food carcinogens, with possible implications for
cancer susceptibility.
Conclusion
Molecular epidemiology has contributed to a growing awareness of the importance of
relatively common genetic and acquired susceptibility factors in modulating risks associated
with exposure to environmental carcinogens. The future challenge of molecular
epidemiology is to analyse individuals who are exposed to carcinogens for a combination of
genotypes associated with susceptibility to cancer. It is evident that use of more precisely
measurable intermediary risk markers, like DNA adducts, cytogenetic damage, and
mutations, rather than cancer as an end point, will allow the identification of combinations
of cancer-relevant genes that positively or negatively affect cancer outcome in humans.
Despite the high number of studies on polymorphisms and cancer susceptibility published in
the last decade, many associations between polymorphisms and common type of cancer
have not been sufficiently investigated, either because the allelic variants have been
described recently, because the number of participants in the study was too small, because
several associations were reported just once, or because discrepant findings do not permit to
obtain conclusions until more information is available. The polymorphisms have been
identified by different genotyping techniques, or in patients with different ethnic origin, that
often show different frequencies for risk alleles, hence comparison of different studies is
difficult. It is intriguing why some variants that are not related to major changes on enzyme
activity or properties are associated with cancer risk.
It is evident that the relationship between any polymorphism locus and disease is best
studied using a very large number of well defined and matched subjects, with confounding
factors, such as smoking and diet, taken into account. The importance of screening more
than one SNP is becoming more important, and made possible due to new technologies
available. The main task, however, will be to characterize the functional significance of these
gene variants in humans.
Any association between polymorphisms in genes for metabolising enzymes and drug
transporters and disease could also result from linkage disequilibrium (LD). LD occurs when
two specific allelic variants located on the same chromosome are combined. Analysis of
linkages and molecular haplotyping techniques, may be needed to characterize haplotypes
in individuals with a highly polymorphic and large gene. Haplotype identification may prove
to be vital in identifying the functional significance of polymorphisms on disease
susceptibility. Besides, determination of haplotypes is important for screening which SNPs
have to be analysed in routine assessment.
Knowledge of the prevalence and distribution of common genetic susceptibility factors and
the ability to identify susceptible individuals or subgroups will have substantial preventive
87
Chapter 1.2
implications, in particular if more data are collected to show that people with certain at
risk genotypes are more susceptible to low levels of exposure. However, before results of
individual screening for genetic traits can be used efficiently to implement preventive
measures, more cancer-predisposing genes need to be studied and gene-environment and
gene-gene interactions elucidated. To this purpose, the need of well-designed, large-scale
studies is emphasized. This review of SNPs in drug metabolising enzymes and drug
transporters contributes to the impression how far research has come to date in order to
predict the risk on cancer.
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Chapter 1

Pharmacogenetic screening in cancer

Pharmacogenetic screening in cancer treatment and cancer risk

Pharmacogenetic screening in cancer treatment and cancer risk

GSTM1, GSTP1, GSTT1

GSTM1, GSTP1, GSTT1

GSTM1, GSTP1, GSTT1

Platinum consisting agents

P-gp, MRP2, BCRP

P-gp, MRP2, BCRP

P-gp, MRP2, BCRP

Pharmacogenetic screening in cancer treatment and cancer risk

Pharmacogenetic screening in cancer treatment and cancer risk

Dihydropyrimidine dehydrogenase (DPD)

Pharmacogenetic screening in cancer treatment and cancer risk

DPD activity and 5-FU toxicity

DPD activity and 5-FU toxicity

Leukopenia and severe mucositis during 5-FU treatment, DPD activity

In combination with GIVS14+1A lethal toxicity during 5-FU treatment*

* Clinical effects referred to more than one genotype

Table 3: Effects of polymorphisms in the DPYD gene

Pharmacogenetic screening in cancer treatment and cancer risk

SN-38/SN-38G 7/7 > 6/7 or 6/6

Reduced AUC SN-38G/SN-38 ratios in combination with *28 or *6

Significant predictor of ANC after irinotecan treatment

Table 4: Clinical effects of SNPs in the UGT1A1 gene

Pharmacogenetic screening in cancer treatment and cancer risk

Glutathione S-Transferase (GST)

Table 6: Clinical effects of SNPs in the GST genes

survival for *B/*B genotype in breast cancer patients treated with

predicted CL of etoposide in patients with mutant allele

Homozygous mutant 3-fold in risk of relapse in childhood ALL

Clinical effects referred to more than one genotype

Table 6: Clinical effects of SNPs in the GST genes (continued)

Pharmacogenetic screening in cancer treatment and cancer risk

Arylamine N-Acetyltransferase (NAT)

Thiopurine methyltransferase (TPMT)

Pharmacogenetic screening in cancer treatment and cancer risk

Pharmacogenetic screening in cancer treatment and cancer risk

Wild-type overall survival and high probability of relapse in AML

Wild-type overall survival and high probability of relapse in AML

Table 8: Effects of SNPs in the P-gp gene

Pharmacogenetic screening in cancer treatment and cancer risk

MRP2 (canalicular-multispecific organic anion transporter cMOAT)

Pharmacogenetic screening in cancer treatment and cancer risk

Breast Cancer Resistance Protein (BCRP)

Pharmacogenetic screening in cancer treatment and cancer risk

Pharmacogenetic screening in cancer treatment and cancer risk

Pharmacogenetic screening in cancer treatment and cancer risk

Pharmacogenetic screening in cancer treatment and cancer risk

Pharmacogenetic screening in cancer treatment and cancer risk

Pharmacogenetic screening in cancer treatment and cancer risk

de Jong FA, Marsh S, Mathijssen RH et al. ABCG2 pharmacogenetics: ethnic differences in

Pharmacogenetic screening in cancer treatment and cancer risk

Table 1: Effects of polymorphisms in the CYP1A1 gene

risk of endometrial cancer

Table 1: Effects of polymorphisms in the CYP1A1 gene (continued)

risk of breast cancer in African-American women smoking > 20 years

Table 1: Effects of polymorphisms in the CYP1A1 gene (continued)

Pharmacogenetic screening in cancer treatment and cancer risk

*1B over represented in prostate cancer

Table 2: Effects of polymorphisms in the CYP3A4 gene

Pharmacogenetic screening in cancer treatment and cancer risk

Reduced AUC SN-38G/SN-38 ratios in combination with 28 or 6

survival for B/B genotype in breast cancer patients treated with