the role of genetic variability in drug metabolism pathways in breast cancer prognosis

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The role of genetic variability in drug metabolism pathways in breast cancer prognosis
Ji-Yeob Choi1, Susan A Nowell2, Javier G Blanco3 & Christine B Ambrosone1†
†Author for correspondence1Roswell Park Cancer Institute, Department of Epidemiology, Elm & Carlton Sts, Buffalo, NY, 14263, USATel.: +1 716 845 1350;Fax: +1 716 845 8125;E-mail: [email protected] of Arkansas for Medical Sciences, Department of Environmental and Occupational Health,4301 W. Markham St,#820, Little Rock, AR 72205, USA3State University of New York, Department of Pharmaceutical Sciences, 517 Hochstetter Hall, Buffalo, New York 14260–1200, USA

Keywords: anthracyclines, aromatase inhibitors, breast cancer, cyclophosphamide, pharmacogenetics, single nucleotide polymorphisms, tamoxifen, taxanes

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Among patients receiving adjuvant therapy for breast cancer, there is variability in treatment outcomes, and it is unclear which patients will receive the most benefit from treatment and which will have better disease-free survival. To date, most studies of breast cancer prognosis have focused on tumor characteristics, but it is likely that pharmacogenetics, genetic variability in the metabolism of therapeutic agents, also plays a role in the prediction of survival. In this paper, we briefly discuss the metabolic pathways of drugs commonly used for the treatment of breast cancer (cyclophosphamide, doxorubicin, taxanes, tamoxifen and aromatase inhibitors) and describe the known genetic variants that may impact those pathways. Studies that have evaluated potential effects of these genetic variants on treatment outcomes are also discussed. It is likely that the application of pharmacogenetics, particularly in the setting of randomized clinical trials, will contribute to findings that may result in individualized therapeutic dosing.

Significant variability in drug response mayoccur among cancer patients treated with thesame medications. The pharmacodynamics ofcurrent anticancer drugs is often unpredictableand may reflect complex interactions betweengenetic and epigenetic factors, resulting in varia-ble therapeutic outcomes. Pharmacogenetics, thestudy of the role of inheritance in interindividualvariability in drug response, has the potential tocontribute to the development of more rationalpharmacological therapies for various types ofcancers. Successful pharmacogenetic strategieshave been developed to individualize the admin-istration of thiopurines within the setting oftherapy for childhood acute lymphoblasticleukemia, and for the use of 5-fluorouracil(5-FU) for colon cancer [1]. The pharmacogenet-ics of breast cancer therapy is also an area ofintensive research, and the role of interindividualdifferences in drug metabolism in relation tobreast cancer outcomes remains to be elucidated.In this review, the drugs commonly used for thetreatment of breast cancer and their metabolicpathways are briefly described, and the potentialrole of genetic variability in those pathways inclinical outcomes is discussed.

Breast cancer therapeuticsThe drugs most commonly used for the treat-ment of breast cancer include cyclophosphamide(C) and doxorubicin (Adriamycin® [A]), withmethotrexate and 5-FU now used less frequently[2]. Treatment with these agents is often followedby the administration of taxanes (T), such aspaclitaxel and docetaxel, which have been shown

to enhance survival benefits over use of CA alone[3]. Among women with estrogen receptor (ER)-positive tumors, tamoxifen use has resulted in a47% reduction in breast cancer recurrence and a26% decrease in overall mortality. Althoughtamoxifen is likely to remain the anti-estrogen ofchoice for some women, particularly premeno-pausal women with ER+ tumors, the third-gen-eration aromatase inhibitors (AIs), such asanastrozole, have been shown to have greaterefficacy than tamoxifen, and have fewer sideeffects [4]. In the sections below, we address thepharmacogenetics of the primary drugs used forbreast cancer treatment, as shown in Table 1, andwhat is known regarding variability in genesinvolved in the metabolic pathways for thosechemotherapeutic agents. Clinical data, if availa-ble, regarding the effects of genetic poly-morphisms upon specific drug metabolismpathways are also presented, and studies evaluat-ing effects of genetic variability on clinicaloutcomes are summarized.

Cyclophosphamide Cyclophosphamide undergoes both Phase I and IImetabolism, with bioactivation through cyto-chrome P450 (CYP) enzymes to 4-hydroxycyclo-phosphamide. This is catalyzed primarily byCYP2B6, CYP3A4, and CYP2C9, with CYP2A6,CYP2C8, and CYP2C19 making minor contri-butions (reviewed in [5]). The 4-hydroxy metabo-lite undergoes a nonenzymatic eliminationreaction to phosphoramide mustard (PM) andacrolein, the metabolites associated with bladdertoxicity. The amount of PM available for cellular

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effects is likely to be influenced by several meta-bolic pathways. The aldehyde dehydrogenasestransform aldophosphamide to carboxyphos-phamide (CP) [6], the major urinary metabolite,which in turn can be dechloroethylated byCYP2B6 and CYP3A4 [7]. It has been suggestedthat variability in CYP2B6 activity could beresponsible for severe toxicities, as CYP2B6 cata-lyzes metabolism of CP to 2- and 3-dechloroe-thyl-chloroacetaldehyde, which is a neurotoxicby-product. Inactivating reactions of cyclo-phosphamide intermediates are catalyzed by glu-tathione S-transferases (GST) A1 and P1 [8]. TheGSTs also participate in protecting cells fromtreatment-generated oxidative stress, along withsuperoxide dismutase (SOD), catalase (CAT) andglutathione peroxidase (GPX1). Thus, theenzymes that appear to be central to the metabo-lism of cyclophosphamide are CYP2B6, CYP3A4,CYP2C9, GSTA1 and GSTP1.

CYP2B6 appears to be involved in the metabo-lism of a number of clinically important drugs,including tamoxifen, and is discussed here in rela-tion to its role in the metabolism of cyclophospha-mide. It is likely that variability in expression andactivity occurs through genetic polymorphisms, aswell as through exposure to enzyme inducers. Anumber of single nucleotide polymorphisms(SNPs) have been identified in CYP2B6, although

only a few have been investigated in relation tofunctional relevance. In in vitro studies of drugmetabolism, the Q172H variant was associatedwith increased 7-ethoxycoumarin-O-deethylaseactivity [9], and the K262R polymorphism wasassociated with an increased Vmax and Vmax/Kmwith 7-ethoxy-4-trifluoromethylcoumarin as asubstrate [10]. Homozygosity for the R487C poly-morphism was associated with an eightfold lowermean protein expression level than from cell lineshomozygous for common alleles [11]. Lang andcolleagues found that four less frequent SNPsencoding for nonsynonymous amino-acid substi-tutions (M46V, G99E, K139E and I391N)resulted in protein variants with very low or nullenzymatic activity [12]. In a small study of patients(n = 29) with hematological malignancies whoreceived a conventional dose of cyclophospha-mide, Xie and colleagues reported that theCYP2B6*6 allele (Q172H and K262R) was asso-ciated with increased 4-hydroxylation of cyclo-phosphamide in human liver, and that the G516Tallele contributed to twice the clearance of thedrug than common alleles [13]. However, in apatient group receiving a number of treatmentagents [14], pharmacokinetic parameters for cyclo-phosphamide did not differ by CYP2B6 geno-type. CYP2B6 is highly inducible, withrelationships between CYP2B6 genotype and

Table 1. Enzymes involved in anticancer drug metabolism.

Enzymes Substrates Effect

CYPs

CYP2B6 Cyclophosphamide Activation

CYP2C8, CYP2C9 Paclitaxel Inactivation

CYP2D6 Tamoxifen Activation

CYP3A4, CYP3A5 CyclophosphamideTamoxifen

ActivationInactivation

Phase II

GSTM1, GSTT1, GSTA1, GSTP1 NonspecificCyclophosphamide

Inactivation

UGTs Nonspecific Inactivation

SULTs Tamoxifen Inactivation

Others

CBR Doxorubicin Inactivation/toxic metabolite

ALDH1A1 Cyclophosphamide Inactivation

MnSOD Nonspecific Increased ROS

CAT Nonspecific Decreased ROS

MPO Nonspecific Increased ROS

ALDH1A1: Aldehyde dehydrogenase 1 family, member A1; CAT: Catalase; CBR: Carbonyl reductase 1; CYP: Cytochrome P450; GSTA1: Glutathione S-transferase A1; GSTP1: Glutathione S-transferase π; GSTM1: Glutathione S-transferase µ1; GSTT1: Glutathione S-transferase θ1; MnSOD: Manganese superoxide dismutase; MPO: Myeloperoxidase; ROS: Reoxygenation; UGT: Uridine diphosphate glycosyltransferase.

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The role of genetic variability in drug metabolism pathways in breast cancer prognosis – REVIEW

CYP2B6 protein expression differing significantlyby gender and ethnicity [15], and by exposuresincluding smoking and alcohol use [16].

The CYP3A subfamily is predominant in thehuman liver, and participates in the metabolismof many clinically used drugs. CYP3A4 is themajor hepatic CYP3A, and is also present in theintestinal epithelium. CYP3A5 is hepatic as wellas extrahepatic, and CYP3A4 and CYP3A5 haveoverlapping substrate specificities [17].

A number of genetic variants have been identi-fied in CYP3A4, many of which result in aminoacid substitutions with effects on catalytic activity.In a pharmacokinetic study of 71 women, it wasnoted that those with the CYP3A4*1B allele had ahigher area under the concentration–time curve(AUC) of the parent drug, indicating less enzymeactivity [14]. However, it is generally accepted thatmost of the known SNPs in the coding and5´-flanking regions of CYP3A4 are not the maindeterminants for the large interindividual variabil-ity of CYP3A4 expression or activity [18–20]. Asreviewed by Schuetz [21], there is growing agree-ment that human variation in CYP3A4 activitymay be caused by polymorphisms alteringCYP3A4 gene regulation, or by factors that induceor inhibit it. For example, it is known that CYP3Aexpression is induced by steroid hormones, someflavonoids and botanicals, polychlorinated biphe-nyls (PCBs) and organochlorine pesticides, mac-rolide antiobiotics, antifungal agents, and receptorand enzyme antagonists [22]. Thus, variability inCYP3A4 expression may be the result of geneticand nongenetic factors other than SNPs in thecoding and promoter regions of the gene.CYP3A5 contains a number of variants; the com-mon variant, CYP3A5*3, results in loss of expres-sion of the enzyme, due to the creation of acryptic splice site, resulting in a truncatedprotein [23]. The CYP3A5*1 variant was assessedin the pharmacokinetic study discussed above,which found that the AUC of cyclophosphamidewas higher in patients with the *1 allele [14]. RareCYP3A5 alleles have been observed in Afri-can–Americans; CYP3A5*6 results in abnormalsplicing, and CYP3A5*7 results in a frameshiftmutation, both of which appear to affect enzymeactivity [24].

The CYP2Cs are responsible for the metabo-lism of approximately 20% of clinically-useddrugs. There are four members of the sub-family, CYP2C8, CYP2C9, CYP2C19 andCYP2C18. Although CYP2C9 and CYP2C19are widely considered to be clinically importantmembers of the CYP2C family, there are fewdata regarding their activity toward anticancer

drugs [25]. In the Xie study noted above,CYP2C9 polymorphisms did not influenceclearance of cyclophosphamide [13].

As described above, cytosolic aldehydedehydrogenases (ALDHs) catalyze the oxidationof aldophosphamide, an intermediate of cyclo-phosphamide activation. This process blocks thegeneration of phosphoramide mustard throughthe formation of inactive carboxyphosphamide.Thus, it has been proposed that increased levels ofALDH might confer resistance to cyclo-phosphamide [26]. Several ALDH1A1 variantsoccur in humans, but have low prevalence inEuropean–Americans (≤0.05) [27]. Kinetic studieshave only evaluated activity in relation to ethanolmetabolism [28].

GSTs are Phase II enzymes that catalyze detoxi-fication conjugations of several chemotherapeuticdrugs or their metabolites. As noted above,GSTP1 and GSTA1 are the primary GSTs thathave activity toward cyclophosphamide metabo-lites. Genetic variability resulting in decreased orabsent enzymatic activity could result in higherlevels of cytotoxic metabolites and in better tumorcell kill and/or increased toxicity.

GSTA1 is the primary hepatic GST, but is alsoexpressed in the human breast [29], and has thegreatest activity toward cyclophosphamide. TheGSTA1*B genetic polymorphism contains threelinked base substitutions in the promoter at posi-tions -567, -69 and -52, respectively [30]. In vitro,the GSTA1*B allele results in lower transcriptionalactivation as compared with the commonGSTA1*A allele, and the G>A change at position-52 is responsible for differential promoter activity,altering binding of the ubiquitous transcriptionfactor SP1 [31].

The enzymatic activity of GSTP1 is affectedby two known genetic polymorphisms, Ile105Valin exon 5 and Ala114Val in exon 6. The GSTP1Val105 variant allele is fairly common (approxi-mately 26% for V105 allele in Caucasians), butthe GSTP1 Val114 variant is infrequent. Cata-lytic efficiency with two alkylating agents (thi-otepa and chlorambucil) was demonstrated to behigher for the common GSTP1 I105, A114 pro-tein isoform compared with either the V105,A114 or V105, V114 variant isoforms [32,33]. Ascyclophosphamide is structurally similar to thi-otepa, patients with GST allelic variants may havedifferential rates of active removal of that drug.

AnthracyclinesThe antitumor activities of anthracycline drugssuch as doxorubicin, daunorubicin and epiru-bicin result from the combination of various

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intracellular effects. The main mechanisms ofanthracycline-mediated tumor cell kill involve:

• DNA topoisomerase II inhibition

• DNA damage through p53-dependent and/orp53-independent mechanisms

• Induction of apoptosis mediated bycytochrome c release

• Proteasome interactions

• Oxidative damage mediated by generation offree radicals (reviewed in [34])

The complex chemical structure of anthra-cyclines allows the formation of several metabo-lites. A number of oxidoreductases (e.g.,microsomal NAD(P)H-cytochrome P450reductase, cytoplasmic NADH-dependent xan-thine dehydrogenase and nitric oxide synthase)catalyze the one electron reduction of the qui-none (Q) moiety. The resulting semiquinones(Q) readily regenerate the parent Q by reducingoxygen to O2¯ and hydrogen peroxide (H2O2).In addition, reductase-type glycosidases andhydrolase-type glycosidases catalyze the forma-tion of a panel of anthracycline aglycone metab-olites. Anthracyclines are also good substrates forcytosolic carbonyl reductases and aldo-ketoreductases. These enzymes catalyze the two-elec-tron reduction of the side chain C-13 carbonylgroup to form more polar anthracycline–alcoholmetabolites (e.g., doxorubicinol and daunorubi-cinol) [35]. Interestingly, several lines of evidenceindicate that anthracycline–alcohol metabolitesplay a key role in the pathogenesis of anthra-cycline-related chronic cardiotoxicity [36,37].Some anthracyclines undergo extensive metabo-lism through Phase II reactions. For example,epirubicin and epirubicinol are conjugated withglucuronic acid by hepatic uridine diphosphate-glucuronosyltransferase (UGT) 2B7 [38]. Varia-bility in a number of pathways could therebyaffect treatment outcomes among patientstreated with anthracyclines, although littleresearch has been conducted in this area. In par-ticular, polymorphisms in the carbonyl reduct-ases could modify cellular effects, as well asvariability in UGT2B7.

The anticancer anthracyclines, doxorubicinand daunorubicin, are extensively reduced bycarbonyl reductases (CBRs) in normal tissues, aswell as in tumors, into their corresponding alco-hol metabolites. In humans, there are two mono-meric carbonyl reductases, carbonyl reductase 1(CBR1) and carbonyl reductase 3 (CBR3),which are encoded by different genes located onchromosome 21 (CBR1 and CBR3) [39]. Variable

cytosolic CBR activities have been documentedin breast and lung tumors, as well as in humanliver [40,41]. Blanco and colleagues have character-ized the functional impact of a common geneticpolymorphism in CBR3 (CBR3 V244M). BothCBR3 isoforms (CBR3 V244 and CBR3 244M)have distinctive catalytic properties toward thequinone menadione and the nicotinamide ade-nine dinucleotide phosphate, reduced form(NADP[H]) cofactor [42]. Studies are underwayto elucidate the impact of these common CBRpolymorphic variants in the variable pharmaco-dynamics of the anticancer anthracyclinesamong breast cancer patients.

There are several SNPs in UGT2B7 that appearto have functional effects on enzyme activity. Inparticular, eight SNPs have been identified in thepromoter region of the gene, six of which aretightly linked, and at least four haplotypes havebeen observed in Caucasians. Gene constructswith the -79 variation displayed 2.5- to sevenfoldless activity compared with the wild-type con-struct in cell lines, and in vivo, serum morphineand morphine glucuronide concentrations variedby genotype [43].

Another mechanism of tumor cell cytotoxicityby anthracyclines is through the induction ofmitochondrial changes and apoptosis through anoxidative stress pathway [44,45], resulting in mas-sive cellular damage associated with lipid peroxi-dation and alterations of proteins and nucleicacids. Apoptosis occurs when, through a path-way of signaling, the mitochondrial membranebecomes permeable [46–48] and while reoxygena-tion (ROS), among other factors, induces orfacilitates mitochondrial permeability, glutath-ione and antioxidant enzymes, such as manga-nese superoxide dismutase (MnSOD), catalase(CAT) and glutathione peroxidase 1 (GPX1),inhibit it [47]. Furthermore, myeloperoxidase(MPO) produces hypochlorous acid from hydro-gen peroxide generated by MnSOD, resulting inhigher levels of ROS. A high ROS environmentresulting from variability in these pathways mayaffect therapeutic outcomes.

MnSOD is synthesized in the cytosol andpost-transcriptionally modified for transportinto the mitochondrion, where it catalyzes thedismutation of two superoxide radicals, produc-ing H2O2 and oxygen. A polymorphism inMnSOD exists in codon 16, which is located atposition -9 of the mature protein and results inthe incorporation of either alanine (C allele) orvaline (T allele) in the mitochondrial targetingsequence. Recent experimental data indicate




that the alanine containing MnSOD is targetedinto the mitochondria, whereas the valine formof the protein is partially arrested in the innermitochondrial membrane [49]. While one wouldintuitively hypothesize that the less efficientform (T) would be associated with higher levelsof ROS and greater risk of cancer, it is the Cpolymorphism that has been associated with riskof breast [50], prostate [51], and bladder [52], butnot lung [53] cancer.

CAT is a heme enzyme that has a predominantrole in controlling H2O2 concentration in humancells, by converting H2O2 into H2O and O2.With MnSOD and GPX1, CAT constitutes aprimary defense against oxidative stress and mayprovide resistance to the effects of chemotherapy,A common polymorphism has been identified inthe promoter region of the CAT gene, a -262 CTsubstitution on the 5´ region of the human CATgene from the transcription start site [54]. In alarge population of healthy controls, it wasrecently shown that the TT genotypes were asso-ciated with reduced catalase activity measured inerythrocytes [55].

A frequently-occurring SNP in the promoterregion of the MPO gene is a -463 GA substitu-tion, which is located in a SP1 consensus bind-ing site [56]. The MPO A variant allele results inlower transcriptional activation as comparedwith the -463 G allele because of the disruptionof the SP1 binding site [57]. In support of theseobservations, the -463 G allele has been associ-ated with increased MPO mRNA and proteinlevels in myeloid leukemia cells [58].

TaxanesTaxanes, such as paclitaxel and docetaxel, exerttheir cytotoxic effects by binding to β-tubulin,followed by the stabilization of microtubules anddisruption of mitotic spindle formation duringcell division, resulting in cell death [59]. Paclitaxelis metabolized in the liver by cytochrome P450enzymes, with extensive conversion to6α-hydroxy-paclitaxel by CYP2C8 [60], and pro-duction of 3´-p-hydroxyphenylpaclitaxel, a lessermetabolite, by CYP3A4/CYP3A5. Both of thesehydroxylated metabolites can undergo furthermetabolism to form the dihydroxy metabolite,6-α-hydroxy-3’-p-hydroxyphenylpaclitaxel [5,60].It appears that docetaxel is metabolized mainlyby CYP3A4 to one major and three minormetabolites, and some laboratory studies indi-cate that CYP3A4 inhibitors could be used toreduce the metabolism of docetaxel, resulting inhigher circulating levels [61].

CYP3A4 and 3A5 are discussed above in thecontext of cyclophosphamide metabolism, andthe points are also relevant for outcomes withtreatment using taxanes. CYP2C8 has beenshown to be involved in the metabolism of pacl-itaxel [60], and there are a number of SNPs in thegene, with CYP2C8*2 (I264F) present in Afri-can–Americans, and CYP2C8*3 (R139K,K399R) occurring only in European–Americans.The functional significance of the CYP2C8 poly-morphisms is unclear. An early in vitro studydemonstrated that the *2 allele was associatedwith 15% of the activity toward paciltaxelhydroxylation of the common alleles [62], andseveral other studies showed that the CYP2C8*3variant exhibited 50% activity toward paclitaxel6α-hydroxylation when compared withCYP2C8*1 alleles in vitro [63–65]. However, morerecent studies have not supported a functionalsignificance for these polymorphisms. In apharmacokinetic study, CYP2C8 genotypes werenot significantly associated with unbound clear-ance of paclitaxel [66]. In a similar study in a Jap-anese population, none of the 23 patients hadany variant SNPs [67].

Anti-estrogensTamoxifen (TAM) is a selective estrogen receptormodulator (SERM) that binds to the ER, pre-venting binding of the endogenous ligand, estra-diol, and inhibiting estradiol-driven cellularproliferation in tumors, although it appears thatTAM may also have nongenomic mechanisms ofaction, influencing several apoptotic pathways(reviewed in [68]).

The pharmacology and metabolism of TAM iscomplex, with the compound undergoing exten-sive Phase I and II metabolism. The metabolitesof TAM exhibit varying degrees of antiestrogenicor estrogenic properties, therefore the relative lev-els of the metabolites and their intrinsic pharma-cological effects combine to produce the overalloutcomes of TAM therapy [69]. Several CYPs par-ticipate in the metabolism of TAM to producethe major metabolite, N-desmethyl tamoxifen.Studies with recombinant enzymes show thatCYPs 3A4, 2C9, 1A1, 1A2 and 2D6 contributeto the formation of this metabolite [69,70]. N-des-methyl tamoxifen has a low affinity for the estro-gen receptor, and, until recently,4-hydroxytamoxifen (4-OH TAM) was consid-ered to be the primary active metabolite of TAM,due to its high affinity for the estrogen receptor.However, recent work by Flockhart and col-leagues demonstrated that 4-hydroxy-N-des-

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methyltamoxifen (endoxifen), is present inplasma at concentrations more than eight timesthat of 4-OH-TAM, and has equal affinity for theestrogen receptor [71]. For this reason, endoxifenmay be the more physiologically relevant activemetabolite [72]. 4-OH TAM is formed primarilyby the action of CYP2D6, although other CYPscan contribute [73,74]. Endoxifen is predominantlyproduced by CYP2D6 [72]. Once formed, 4-OHTAM and endoxifen are also subject to Phase IIconjugation reactions, with preferential sulfationof trans-4-OH TAM by sulfotransferase family,cytosolic 1A, phenol-preferring member 1(SULT1A1), and glucuronidation of cis-4-OHTAM by UGT2B15 [75].

CYP2D6 is involved in the metabolism ofnumerous drugs, and was first identified for itsphenotypic effects on drug metabolism. To date,46 allelic variants have been reported, and theycan be classified based upon their effects onenzyme activity, either to increase, decrease ortotally eliminate CYP2D6 activity [76]. Most ofthe variation in CYP2D6 activity is explained bya subgroup of CYP2D6 SNPs; severe drug reac-tions occur in CYP2D6 European–Americanpoor metabolizers, 95% of whom can be identi-fied by genotyping for CYP2D6*3, CYP2D6*4,CYP2D6*5 and CYP2D6*6 alleles [77]. Conse-quently, these CYP2D6 alleles are the focus ofmost pharmacogenetic studies.

The UGTs participate in the detoxification ofa number of substrates, through catalyzing trans-fer of the glucuronyl moiety from uridine5´-diphosphoglucuronic acid to functionalgroups of a variety of compounds, facilitatingtheir excretion. UGT2B15 catalyzes thebiotransformation of a number of steroid sub-strates, including 4-OH TAM [78]. TheUGT2B15*2 (Asp85Tyr) polymorphism pro-duces an amino acid change at residue 85 fromaspartate (D85) to tyrosine (Y85). The variant islocated within the putative substrate recognitionsite of the enzyme and is associated with anincreased velocity of reaction [79]. UGT2B15*2variants appear to be a risk factor for the recur-rence and poorer survival of breast cancerpatients treated with tamoxifen who also havethe variant SULT1A1*2 allele (Arg213His) [80].

Among ten SULT forms, potentially impor-tant polymorphisms are known for SULT1A1(phenol sulfotransferase), SULT1A2, andSULT2A1 (hydroxysteroid or dehydroepiandros-terone sulfotransferase). The variant alleleSULT1A1*2 (Arg213His) has been associatedwith reduced enzymatic activity. When overall

survival of breast cancer patients was evaluatedaccording to SULT1A1 genotype, Nowell andcolleagues found that individuals homozygousfor the variant allele had a threefold increase inrisk of death compared with patients with one ormore common alleles [81]. Wegman and col-leagues reported similar associations betweenSULT1A1 genotype and survival among patientstreated with TAM [82]. The results of these stud-ies can be difficult to interpret, in that highSULT1A1 activity would theoretically lead tomore rapid elimination of active metabolites.However, a recent abstract presented at theAmerican Association for Cancer Research(AACR) indicates that the sulphated metaboliteof 4-OH TAM possesses toxic properties [83].The variant allele of SULT1A2*2 (Asn235Thr)reduces the affinity of the enzyme for its sub-strates [84], and is reported to be in linkagedisequilibrium with SULT1A1*2 [85].

Aromatase inhibitorsAromatase inhibitors (AI) prevent the conver-sion of androgens to estrogens by inhibiting theactivity of cytochrome P450 aromatase(CYP19). Aromatase inhibition is a useful thera-peutic approach to treat estrogen-dependentbreast cancers, particularly in postmenopausalwomen. The AI, anastrazole, appears to beextensively metabolized through N-dealkylation,hydroxylation and glucuronidation, consistentwith the involvement of the cytochrome P450enzyme system. The known metabolites of anas-trozole are triazole, a glucuronide conjugate ofhydroxyl–anastrozole, and a glucuronide of anas-trozole itself [86]. To our knowledge, the quanti-tative contribution of the formation of each ofthese metabolites and the specific enzymesinvolved are not known.

Clinical & molecular epidemiological studies of pharmacogenetics & treatment outcomesAs shown in Table 2, most studies of pharmaco-genetics and breast cancer have been conductedamong patients receiving heterogeneous treat-ments, usually consisting of cyclophosphamidealong with other drugs. The majority of thesestudies have focused on the Phase II enzymepathways. In a study of 92 women withadvanced breast cancer, Lizard-Nacol and col-leagues [87] found no association betweenGSTM1-null genotype and prognostic factors,clinical response rate (complete and partialresponses), or overall survival. Ambrosone and




Table 2. Association

Genes SN

Cyclophosphamide (C

GSTM1 Gge

GSTP1 G

GSTA1 G

CYP3A4; CYP3A5; GSTM1/T1

CCGge

17 genes i.e., CYPs; GSTs; ABCB1

29

Tamoxifen (TAM)

SULT1A1 SU

CYP2D6; CYP2C9;CYP3A5;SULT1A1

C*5CCSU

CYP2D6; UGT2B15; SULT1A1

CUSU

CYP2D6; CYP3A5 CC

Oxidative stress

GSTM1/T1 Gge

MnSOD; CAT; MPO MA-2-4

*Events include death for oABCB1: Adenosine triphospCI: Confidence interval; CP:GSTM1: Glutathione S-tranMPO: Myeloperoxidase; OScytosolic, 1A, phenol-prefe

Sweeney evaluated variability in several membersof the GST family, including GSTA1 andGSTP1 in relation to cyclophosphamide metab-olism, and GSTM1 and GSTT1 for response tooxidative stress generated by therapeutic agents.As hypothesized, they found that womenhomozygous for genotypes associated with loweractivity of GSTP1 Val105 or GSTA1*B (C-69T)

had better overall survival, with risk of deathreduced to 30–50% in a retrospective study of240 women, 97% of whom received cyclo-phosphamide [88,89]. They also found thatwomen with GSTM1 and GSTT1 gene deletionshad better survival than those with allelespresent; women who had null genotypes forboth GSTs had a two-thirds reduction in risk of

studies of chemotherapy and genetic polymorphisms in drug-metabolizing enzymes.

P Subjects* Treatment End point Risk (95% CI) Ref.

P)

STM1 null notype

92 total patients CAF OS or DFS GSTM1 not significant [87]

STP1 Ile105Val 71/240 events CP-based OS GSTP1 Val/Val HR = 0.3 (0.1–1.0)

[88]

STA1*B 66/245 events CP-based 5-year survival GSTA1*B/*B HR = 0.5 (0.3–1.0)

[89]

YP3A4*1B; YP3A5*3; STM1/T1 null notype

35/90 events Anthracycline-based with CP

OS or DFS Low-drug group (CYP3A4*1B- CYP3A5*3-GSTs present) vs high-drug group HR = 4.8 (1.8–12.9) for OS

[91]

SNPs 55/85 events CP and cisplatin

OS and PK of CP Each CYP3A4*1A, CYP3A5*3, GSTM1 null better OS (p < 0.05)

[14]

LT1A1*2 100/337 events TAM OS SULT1A1*2/*2 HR = 2.9 (1.1–7.6) with TAM treated only

[81]

YP2D6*3, *4, , *6;

YP2C9*2, *3; YP3A5*3;

LT1A1*2

80 total patients TAM w or w/o CYP2D6 inhibitor

Plasma concentration of TAM and its metabolites

CYP2D6*Vt/Vt decreased plasma concentration of endoxifen (p < 0.001)

[93]

YP2D6*4; GT2B15*2;LT1A1*2

106/337 events TAM OS CYP2D6 not significant; UGT2B15*2 and SULT1A1*2 HR = 4.4 (1.17–16.55) for OS with TAM treated only

[80]

YP2D6*4, *6; YP3A5*3

101/233 events TAM OS or DFS CYP2D6*4/*4 HR = 1.9 (0.91–3.82) for DFS

[94]

STM1/T1 null notype

74/251 events Combination OS or DFS GSTM1/T1 both null genotype HR = 0.3 (0.11–0.70) for OS

[90]

nSOD la-9Val; CAT 62C>T; MPO 63G>A

83/279 events Combination OS MPO G allele HR = 0.6 (0.38–0.95)Both MPO GG & MnSOD CC HR = 0.3 (0.13–0.80)

[92]

verall survival or recurrence for disease-free survival.hate-binding cassette, subfamily B, member 1; CAF: Cyclophosphamide; doxorubicin and 5-flurouracil; CAT: Catalase;

Cyclophosphamide; DFS: Disease-free survival; GSTA1: Glutathione S-transferase A1; GSTP1: Glutathione S-transferase π; sferase µ1; GSTT1: Glutathione S-transferase θ1; HR: Hazard ratio; MnSOD: Manganese superoxide dismutase; : Overall survival; PK: Pharmacokinetics; SNP: Single nucleotide polymorphism; SULT1A1: Sulfotransferase family, rring, member 1; TAM: Tamoxifen; UGT: Uridine diphosphate glucuronosyltransferase.

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death [90]. In the Petros study, survival time wasassociated with GSTM1 genotypes, with patientswith null alleles having better survival [14].GSTM1 null alleles were also associated withbetter survival in a study of 90 breast cancerpatients [91].

Few studies have evaluated the impact of poly-morphisms in Phase I metabolism of cyclo-phosphamide or paclitaxel. In the Petros study ofpatients treated with cyclophosphamide, cispla-tin and carmustine, improved survival occurredfor those with CYP3A4*1B, and for those withCYP3A5*1 genotypes, although the sample sizewas small (n = 85) [14]. In a study of a similar size(n = 90), patients with CYP3A4 *1B genotypesor CYP3A5*1 alleles also had better survival thanthose with variant genotypes, although numbersof patients with *B and *3 genotypes were low,and associations were not significant [91]. Whengenotypes were combined into those likely tohave high, intermediate or low levels of drugreaching the tumor, those with low-drug geno-type combinations had poorer overall survivalthan those with intermediate or high-drug geno-types. To our knowledge, there have been nostudies published evaluating the effects of geneticvariability on treatment outcomes among breastcancer patients receiving taxanes.

The role of variants in genes related to oxida-tive stress, such as MnSOD, CAT and MPO,have also been examined in breast cancer sur-vival. As mitochondrial MnSOD catalyzes con-version of superoxide radicals to H2O2, withcatalase neutralizing H2O2 and myeloperoxidaseconverting H2O2 to highly reactive hypochlo-rous acid (HOCl), gene variants could impactthe efficacy of treatment for breast cancer andimprove survival. In a study by Ambrosone andcolleagues among 298 women who had receivedchemotherapy, women who were homozygousfor the MPO G allele, associated with increasedgene transcription, had better survival than thosepatients with the common AA allele (HR = 0.60,95% CI = 0.38–0.95) [92]. MnSOD C alleleswere associated with nonsignificant, reducedhazard of death, and there were no significantassociations between CAT genotypes and sur-vival. When genotypes associated with higherlevels of ROS for MnSOD and MPO were com-bined, women with MnSOD CC and MPO GGgenotypes had a threefold decrease in hazard ofdeath (HR = 0.33, 95% CI = 0.13–0.80).

As this population was extremely hetero-geneous, the authors further evaluated thesehypotheses in a cohort of women who were

treated on a therapeutic trial (SWOG 8897) andrandomized to CAF or CMF. A portion ofwomen with ‘low-risk’ disease received no adju-vant chemotherapy. Preliminary analyses ofthese data show similar associations to thoseobserved in the previous study between MPOgenotypes and breast cancer survival, with a sug-gestion of effects for MnSOD genotypes as well(unpublished data).

Outcomes among women treated withtamoxifen have been studied in relation to bothPhase I and Phase II enzymatic pathways. In aprospective study of 80 ER-positive womentreated with tamoxifen, plasma levels ofendoxifen were significantly lower in womenwith CYP2D6 homozygous variant genotypes(*3, *4, *5 and *6). Genetic variations inCYP2C9, CYP3A5, or SULT1A1 were not asso-ciated with significant differences in the plasmaconcentrations of tamoxifen or itsmetabolites [93]. Recently, Goetz and colleaguesdemonstrated that women with CYP2D6*4homozygous genotypes had shorter relapse-freetime and poorer disease-free survival comparedwith women with other CYP2D6 alleles in223 breast cancer patients treated withtamoxifen, although results were only significantin univariate analysis [94]. Furthermore, the sam-ple size was small and underpowered to detecttrue associations, given that the frequency of the*4 allele is approximately 7%, and more than250 patients should be examined to drawmeaningful conclusions.

Nowell and colleagues previously evaluatedthe potential effects of SULT1A1*2, associatedwith reduced enzymatic activity, on breast cancersurvival in a retrospective study of 160 patientswho received tamoxifen and 177 untreatedwomen [81]. Among women on tamoxifen, thosewith the SULT1A1*2 homozygous genotype hada threefold increase in hazard of death comparedwith patients with one or more common alleles.There were no observed effects among womennot treated with tamoxifen. The UGT,UGT2B15, is another Phase II detoxificationenzyme that participates in the biotransforma-tion of tamoxifen metabolites. In the same studypopulation, tamoxifen-treated patients withUGT2B15 high-activity genotypes andSULT1A1 low-activity genotypes had increasedrisk of recurrence and poorer survival, althoughthere was no association when investigatingUGT2B15*2 alleles alone. In that study, therewas no significant association betweenCYP2D6*4 allele and overall survival [80].




Highlights

• There is wide variabilicancer patients; not a

• Breast cancer is most Adriamycin® (A), andaromatase inhibitors (receptor (ER).

• Enzymes involved in tP450 (CYP)2B6, CYP3S-transferase (GST)A1to a lesser degree; uricarbonyl reductases (Cresistance 1 (MDR1 [Tcytosolic 1A (SULT1A1

• Oxidative stress is a cyis regulated by enzym(CAT), glutathione pe

• Genes encoding enzypolymorphisms have benzyme activity.

• There are limited dataoutcomes, with somereduced or lower acti

• MPO genotypes relateassociated with bette

• Variants in CYP2D6 an• Future studies, prefer

should be designed todrug metabolism.

• Pharmacogenetics, coas well as patient lifesprediction of outcome

Expert commentaryThe role of pharmacogenetics in the prediction oftherapeutic outcomes among breast cancerpatients is clearly in its infancy, and rigorous stud-ies, with adequate sample size, need to be con-ducted in order to further elucidate relationships

that may be of clinical relevance. These studiescan best be achieved in the context of large coop-erative group clinical trials, wherein patients arereceiving fairly homogeneous, well-documentedtreatment agents, and are carefully followed fortoxicities as well as recurrence status.

Pharmacogenomics is likely to be only onefactor that may influence breast cancer treatmentoutcomes, and future studies should evaluate theinfluence of disease characteristics, patient co-morbidities, and other patient characteristics inrelation to prognosis. While risk factors for can-cer etiology have been well studied, there are fewsound research studies to guide patients and cli-nicians in adoption of behaviors (diet, physicalactivity, supplement use, and so on) that couldmodify risk of recurrence and death, and thesefactors require further investigation.

OutlookThe studies described, to date, have evaluatedthe role of single SNPs and some combinationsof SNPs, in drug metabolism pathways in rela-tion to treatment outcomes. These studies willlikely be strengthened in the future by a morethorough assessment of genetic variability,through the application of haplotype analysisand pathway analysis. It is also possible thatmeasurement of variability across the genome,using whole-genome scans, will result in predic-tive models for treatment outcomes. Assess-ment of genetic variability on a comprehensivemanner, coupled with investigation of diseaseand patient characteristics, as well as lifestylehabits, in prospective studies, will likely yieldimportant results regarding factors that maydetermine outcomes following treatment forbreast cancer.

ty in rates of recurrence and mortality in breast ll of which is explained by disease characteristics.commonly treated with cyclophosphamide (C), taxanes (T), with anti-estrogens (TAM) and/or AI) used in women with tumors positive for estrogen

he metabolism of these agents include: cytochrome A4, aldehyde dehydrogenase (ALDH), glutathione , GSTP1 (C) CYP2A6, CYP2C8, CYP2C9, CYP2C19 dine diphosphate glucuronosyltransferases (UGTs), BRs [A]); CYP2C8, CYP3A4, CYP3A5, multidrug

]); CYP3A4, CYP2D6, sulfotransferase family, ), UGTs (TAM); and UGTs (AI).totoxic mechanism for many treatment agents, and es including superoxide dismutase (SOD), catalase roxidase (GPX1) and myeloperoxidase (MPO).mes in these pathways are polymorphic, and most een associated with functional effects on

on the effects of polymorphisms on treatment findings that women with GST alleles encoding vity have better survival.d to higher levels of reactive oxygen species are

r survival.d SULT1A1 impact efficacy of tamoxifen treatment.

ably conducted in the context of large clinical trials, evaluate genes involved in complex pathways of

mbined with an assessment of tumor characteristics, tyle habits, may provide comprehensive models for s among breast cancer patients.

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the role of genetic variability in drug metabolism pathways in breast cancer prognosis

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