Introduction
Anthracyclines are commonly used for neoadjuvant or adjuvant chemotherapy of locally advanced breast cancer [
1,
2]. They act by inhibition of topoisomerase II alpha and generation of reactive oxygen species eventually resulting in cell cycle arrest and apoptosis [
3,
4]. Epirubicin is a semisynthetic derivative of doxorubicin preferentially used in breast cancer treatment. It is extensively metabolized in the liver to epirubicinol by aldo-keto reductase and in addition aglycones of epirubicin and epirubicinol are formed [
5]. The major inactivation pathway for epirubicin and epirubicinol is glucuronidation catalysed by UDP-glucuronosyltransferases (UGT). Studies using human liver microsomes, expressing UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7 and UGT2B15 revealed that UGT2B7 uniquely converts epirubicin to its glucuronide [
6]. In addition, also the active metabolites of tamoxifen are eliminated by UGT2B7 mediated glucuronidation [
7].
The
UGT2B7 gene is polymorphic with a frequent non-synonymous variant 802 C > T leading to a histidine to tyrosine substitution in codon 268 (His268Tyr). The functional impact of this polymorphism is unclear as studies have shown lower [
8‐
12], similar [
13‐
15], and even higher enzyme activity of the UGT2B7
268Tyr isoform [
16‐
18]. Two
in vitro studies showed no impact of the
UGT2B7
His268Tyr
genotype on the epirubicin glucuronide formation [
6,
19]. However, the presently available
in vitro studies cannot finally clarify, if variants linked with the
UGT2B7
His268Tyr
polymorphism may modulate up- or downregulation of the enzyme under the conditions of adjuvant or neoadjuvant chemotherapy and
in vitro models can, of course, never fully resemble the
in vivo conditions [
19‐
22].
UGT2B7 haplotype analysis revealed six promoter variants (-1306 G > A, -1299C > T, -1112C > T, -900A > G, -327G > A and -161C > T) which modulate UGT2B7 promoter activity and are in perfect linkage disequilibrium with the
UGT2B7
His268Tyr
variant [
19,
20,
22,
23]. In contrast, for 4-hydroxy-tamoxifen and endoxifen, the active metabolites of tamoxifen, a decreased glucuronidation activity was shown for the UGT2B7
268Tyr isoform
in vitro [
9].
Here we present a pharmacogenetic (PGt) analysis of the Austrian
Tumor of breast tissue:
Incidence,
Genetics, and
Environmental
Risk factors (
TIGER) study [
24,
25] to explore the effect of the
UGT2B7
His268Tyr
polymorphism on invasive disease-free survival after epirubicin treatment.
Discussion
Genetic polymorphisms that might be relevant to drug metabolism and elimination have extensively been studied over the last years to explain between-subject variation in drug responses and to better target corresponding treatments [
29‐
32]. Particularly in breast cancer, genetic polymorphisms of proteins involved in drug transport or metabolism have been shown to affect the efficacy of agents such as tamoxifen, taxanes or aromatase inhibitors [
7,
33,
34]. A considerable variation in the response to epirubicin-based chemotherapies has been reported as well [
35‐
37].
The formation of epirubicin glucuronide represent the main inactivating pathway for epirubicin and studies in human liver microsomes expressing a multitude of UGT isoenzymes, revealed that UGT2B7 uniquely converts epirubicin to its glucuronide [
6]. To the best of our knowledge, this is the first study providing evidence that genetic
UGT2B7 variation contributes to these clinical observations. In our analysis, epirubicin-treated women with the
UGT2B7
268Tyr/Tyr
genotype showed a significantly better invasive disease-free survival as compared to carriers of at least one
UGT2B7
268His
allele. As the use of epirubicin is quite common and the difference in the average invasive disease-free survival times between both variant carriers amounted to about one year, we think this finding might be of broad interest and high relevance in cancer therapy, although it still needs to be confirmed.
A lower glucuronidation capacity of the UGT2B7
268Tyr isoform may delay elimination and cause higher exposure to epirubicin and its active metabolites. This may eventually result in the observed longer invasive disease-free survival of carriers of the
UGT2B7
268Tyr/Tyr
genotype. This is in line with biochemical studies revealing a decreased activity of the UGT2B7
268Tyr isoforms assessing other UGT2B7 substrates [
8‐
12]. However, those were contradicted by others [
13‐
18] and by two
in vitro studies showing no impact of the
UGT2B7
His268Tyr
genotype on the epirubicin glucuronide formation [
6,
19]. One of these studies utilized two lines of HK293 cells expressing the two isoforms generated via site-directed mutagenesis [
6]. This methodology implies the disadvantage that the variant leading to an amino acid exchange is solely studied, whereas potentially linked variants remain unconsidered [
6,
16]. This seems to be important, since the
UGT2B7
His268Tyr
variant appears in frequent haplotypes with other variants, especially promoter variants (-1306 G > A, -1299C > T, -1112 C > T, -900 A > G, -327 G > A and -161 C > T) which seem to influence the expression of the two UGT2B7
His268Tyr isoforms [
13,
19‐
22]. The variant alleles of those promoter variants were associated with a two-fold decreased promoter activity and are perfectly linked with the
UGT2B7
268Tyr
allele [
22]. Djebli
et al. genotyped the -900 A > G promoter variant beside
UGT2B7
His268Tyr
and they reported that all individuals carrying the -900GG genotype carried also two variant
UGT2B7
268Tyr
alleles and those who were heterozygote for the promoter variant were as well heterozygote for
UGT2B7
His268Tyr
[
21]. Using the tagging algorithm implemented in Haploview and the genotype frequencies for the HapMap CEU samples we observed that
UGT2B7
His268Tyr
and the linked promoter variants captured 83% of all genotyped markers (applying a r
2 cut-off of 0.8, minor allele frequency > 0.05, and using NM_001074 +/- 10 kb) [
38,
39]. The
UGT2B7
His268Tyr
variant is further linked to the rather rare promoter variant -138 G > A (less than 2% in Caucasians) which was associated with a seven-fold decreased promoter activity and it cannot be excluded if this variant contributes to our observations [
22]. The presently available
in vitro studies cannot finally clarify, if those promoter variants may modulate up- or downregulation of the enzyme, especially under the conditions of adjuvant or neoadjuvant chemotherapy. Therefore, it remains unclear if our observations can be mainly attributed to the
UGT2B7
His268Tyr
variant or to the expressional regulation caused by linked promoter variants. However, in human liver microsomes, genotyped for
UGT2B7
His268Tyr
, no difference related to this polymorphism in epirubicin glucuronide formation was observed [
19], but it is conceivable that there is an up- or downregulation of UGT2B7 caused by anti-cancer drug therapy which may differ inter-individually depending on genetic variants. Whether or not this does play a relevant role in epirubicin treatment cannot finally be clarified by our study but may be an interesting topic for further research. Sawyer
et al. also observed differences in morphine glucuronidation studying the linked
UGT2B7 -161 C > T variant in pain patients, but failed to confirm their finding in genotyped microsomes [
18]. This may indicate differences between the
in vitro and the
in vivo impact of the variant or the associated
UGT2B7 haplotypes.
A few studies have investigated the impact of the
UGT2B7
268Tyr
variant on the
in vivo metabolism of other drugs. For mycophenolic acid (MPA), a lower glucuronidation activity and MPA-acyl-glucuronide formation rate through the
UGT2B7
268Tyr
variant was shown [
21,
40], which resulted in fewer gastrointestinal side-effects among MPA treated patients [
41]. However, in contrast to MPA-acyl-glucuronide [
41,
42], the glucuronide of epirubicin are not known to contribute considerably to the side effects of the parental drug. Moreover, changes in drug tolerability may affect efficacy outcomes only if those result in changes in treatment adherence. Actually, we have neither data on epirubicin dose modifications and premature discontinuations nor on the incidence of adverse drug reactions. Additionally, an association of the linked
UGT2B7 -161 C > T variant with pharmacokinetics of lamotrigine in epileptic patients has been recently reported [
43]. By contrast, no effect was observed either on pharmacokinetics of efavirenz or zidovudine in human immunodeficiency virus infected patients [
44,
45], or on the treatment response to various opioids [
46‐
48]. Given that our study was a retrospective analysis, we unfortunately were not able to determine plasma levels of drug compounds or metabolites. A possible influencing factor might be the younger age among
UGT2B7
268Tyr/Tyr
carriers in the epirubicin treated patients group, but although age is a relevant covariate for survival, the relatively small difference in mean age between the different
UGT2B7 genotypes is very unlikely to explain a major part of the relatively strong UGT2B7 effects on survival after epirubicin treatment. In addition, we observed no difference in patient age comparing the
UGT2B7 genotype groups within the other patient subgroups and no association between age at diagnosis and outcome in the multivariate analysis. Besides, a link to breast cancer risk has also been discussed for the
UGT2B7
His268Tyr
polymorphism, but no clear evidence has been reported and it is very unlikely that this is a contributing factor to our observations [
49,
50].
In addition, the association of the
UGT2B7
268Tyr
variant with invasive disease-free survival was even more pronounced in the subgroup of epirubicin-treated women who subsequently received tamoxifen (Figure
2b). Actually,
in vitro data suggested reduced glucuronidation rates of the active metabolites 4-hydroxy tamoxifen and endoxifen through the UGT2B7
268Tyr isoform [
9], but in contrast to epirubicin, sulfotransferases also play a quantitatively important role [
51]. Accordingly, no difference in steady state plasma concentrations of tamoxifen metabolites was observed in 240 breast cancer patients receiving 20 mg tamoxifen daily [
52]. In the present study, the sequential treatment with two UGT2B7 substrates resulted in a considerable effect of the
UGT2B7
His268Tyr
variant, whereas there was no difference in invasive disease-free survival among patients not treated with epirubicin, including the subgroup of women treated with tamoxifen alone. The more pronounced effect of the
UGT2B7
His268Tyr
polymorphism in patients treated with both epirubicin and tamoxifen remains currently unexplained and is limited by the rather small sample size of this patient subgroup.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JCS, WR and EHB conceived the idea for the present analysis and designed the study. WR provided the study material. SP, JCS, AHW, UL, WR, PK and EHB collected the data. SP, JCS, AK, JB, AHW, UL, WR, PK and EHB analyzed and interpreted the data. SP, JCS and EHB prepared the manuscript. All authors revised the manuscript and gave their final approval.