This article was downloaded by:[CDL Journals Account][CDL Journals Account]

On: 20 May 2007Access Details: [subscription number 770849124]Publisher: Informa HealthcareInforma Ltd Registered in England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Cancer InvestigationPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713597231

Pharmacogenomics of Colorectal Cancer Preventionand TreatmentHoa Nguyen a; Ashley Tran a; Steven Lipkin a; John P. Fruehauf aa University of California Irvine Chao Family Comprehensive Cancer Center.Orange, California. USA

To cite this Article: Nguyen, Hoa, Tran, Ashley, Lipkin, Steven and Fruehauf, JohnP. , 'Pharmacogenomics of Colorectal Cancer Prevention and Treatment', CancerInvestigation, 24:6, 630 - 639To link to this article: DOI: 10.1080/07357900600896281URL: http://dx.doi.org/10.1080/07357900600896281

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf

This article maybe used for research, teaching and private study purposes. Any substantial or systematic reproduction,re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expresslyforbidden.

The publisher does not give any warranty express or implied or make any representation that the contents will becomplete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should beindependently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings,demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with orarising out of the use of this material.

© Taylor and Francis 2007

Dow

nloa

ded

By: [

CD

L Jo

urna

ls A

ccou

nt] A

t: 23

:23

20 M

ay 2

007

Cancer Investigation, 24:630–639, 2006ISSN: 0735-7907 print / 1532-4192 onlineCopyright c© Informa HealthcareDOI: 10.1080/07357900600896281

CLINICAL SCIENCE REVIEW

Pharmacogenomics of Colorectal CancerPrevention and Treatment

Hoa Nguyen, M.D., Ashley Tran, Pharm.D., Steven Lipkin, M.D., PhD., and John P. Fruehauf, M.D., Ph.D.

University of California Irvine Chao Family Comprehensive Cancer Center, Orange, California, USA.

ABSTRACT

Pharmacogenomic tools are beginning to emerge that will provide guidance in the treat-ment and prevention of colorectal cancer. Significant individual genetic variation exists in drugmetabolism of 5FU, capecitabine, irinotecan, and oxaliplatin that influences both the toxicity andefficacy of these agents. Recent FDA approval of genetic testing for mutations in the UGT1A1gene that predict adverse reactions to irinotecan is ushering in a new era that will increasinglyrely on genotyping to individualize treatment decisions for patients with cancer as well as forpatients at high risk who may be candidates for chemoprevention agents. This review focuseson current knowledge regarding key mutations and polymorphisms which affect outcomes forcolorectal cancer patients, as well as the pharmacogenetics of chemoprevention trials.

INTRODUCTION

Colorectal cancer (CRC) is the second leading killer of allcancers in the United States, affecting more than 150,000 pa-tients annually. Although progress has been made with therecent approval of new agents for this disease, including be-vacizumab, capecitabine, and cetuximab, 5-year survival forpatients with metastatic disease remains less than 15 percent(1). Single agent therapy with 5-fluorouracil has been aug-mented by the addition of oxaliplatin, irinotecan, and mono-clonal antibodies. However, the use of drug combinations andmultiple lines of treatment results in increased toxicity. It istherefore relevant for the clinician to understand the molecularunderpinnings of both drug action and toxicity. Chemotherapydrugs have narrow therapeutic windows and significant poten-tial for harm. Most drugs proven safe by extensive clinical test-ing may be life threatening for the small minority of patients

Keywords: Single nucleotide polymorphism, Pharmacogenetics,Thymidylate synthase, Nucleotide excision repair,Carboxylesterase.Correspondence to:John P. Fruehauf, M.D, Ph.D.UC Irvine Chao Family Comprehensive Cancer Center101 The City Drive, Bld 55, R. 324Orange, CA 92868USAemail: jfruehau@uci.edu

who experience genetically determined idiosyncratic reactions.The use of genetic information to explain interindividual dif-ferences in drug response is referred to as pharmacogenetics(e.g., toxicity, age, comorbidities) and pharmacogenomics (e.g.,resistance/activity, gene expression). Such differences are re-lated in part to polymorphisms in the genes involved in themetabolism of chemotherapy drugs or their receptors, and canhave major contributions to patient outcome (1–3). Knowledgeof how genetic variation influences the effectiveness and toxic-ity of chemotherapy drugs commonly used to treat colorectalcancer supports the role of the physician to enhance patientoutcomes. It long has been recognized that the same medica-tions cause different responses in different patients. Large differ-ences in drug response between human populations, combinedwith small intrapatient variability, suggest a genetic contribu-tion to drug disposition and effects with estimates ranging from20 percent to 90 percent (4). Genetic variation in both drug tar-gets and genes affecting target signal transduction can have aprofound effect on drug efficacy and toxicity, with ≥25 knownexamples (4). These include variants in genes relevant to boththerapeutics and prevention.

Single Nucleotide Polymorphisms (SNPs; pronounced“snips”) are the most common known form of genetic variation,and are estimated to account for greater than 90 percent of thisvariation in humans (5–6). Current human genomic variation isthe result of ∼50,000–100,000 years of evolution that stemmedfrom many different genomic and population events, with evo-lutionary selection occurring with punctuated equilibrium (such

630

Dow

nloa

ded

By: [

CD

L Jo

urna

ls A

ccou

nt] A

t: 23

:23

20 M

ay 2

007

Table 1. Enzymes that exhibit interindividual variations relevant to toxicity or response

Enzyme/Gene name Drug affected Key mutation/polymorphismDihydropyrimidine dehydrogenase (31) 5FU and capecitabine metabolism IVS14 + 1GDPYD A mutationUDP-glucuronosyltransferase 1A1 (42) Irinotecan metabolism T3279GUGT1A1 UGT1A1*28Glutathione-S-transferases (52) Oxaliplatin metabolism A342GGSTP1Thymidylate synthase (27, 34) 5FU and capecitabine activity TS-3′UTRTYMS TS-5′UTRThymidine phosphorylase (35) Capecitabine activationECGF1Excision repair cross-complementing 1 (50) Oxaliplatin sensitivity ERCC1-118 C/TERCC1Excision repair cross-complementing 2 (50) Oxaliplatin sensitivity XPD-751 Lys751LysXeroderma pigmentosum group D ERCC2/XPD

as hypothesized to occur during the last ice age ∼20,000 yearsago). Most of this variation has no obvious pathological or med-ical significance. Polymorphisms are distinguished from mu-tations by an arbitrary frequency criterion: Different forms ofpolymorphism (termed “alleles”) are observed more often in thegeneral population than mutations, with a population frequencyof greater than one percent often used as a cutoff value (7–8).SNPs almost always have a major (more frequent) and minor(less frequent) allele. These are caused by a substitution of onebase for another. The most common type of SNP is a transition:purines are replaced by purines or pyrimidines by pyrimidines(e.g., A to G or C to T). Because humans are diploid for eachSNP, a person can have one of several genotypes: homozygousfor the major allele, heterozygous, or homozygous for the minorallele (e.g., AA, Aa or aa).

Genetic polymorphisms influence drug metabolism predom-inantly due to altered substrate binding affinities that cause in-creased or decreased rates of enzyme activity. Polymorphismsalso can influence protein-protein interactions and enzyme sta-bility. For example, thiopurine S-methyltransferase (TPMT)catalyzes the S-methylation of thiopurine drugs, such as 6-mercaptopurine used on the treatment of acute lymphoblasticleukemia (9). TPMT genetic polymorphisms dramatically in-fluence TPMT activity. TPMT*3A, the most common variantallele for low activity in Caucasians (5 percent), encodes a pro-tein with two altered amino acid residues that cause the proteinto form aggresomes in cells, rendering the enzyme inactive (10).The inactivity and short half life of TPMT in TPMT*3A carriersresults in greater toxicity for patients treated with 6MP.

Other sources of genetic diversity that impact pharmacoge-netics include tandem and dinucleotide repeats. Tandemly re-peated DNA sequences are widespread throughout the humangenome and show sufficient variability among individuals in apopulation that they have become important in genetic mapping,linkage analysis, and human identity testing. These tandemlyrepeated regions of DNA typically are classified into severalgroups depending on the size of the repeat region. Minisatel-lites (variable number of tandem repeats, VNTRs) have core re-peats with 9–80 bp, while microsatellites (short tandem repeats,

STRs) contain 2–5 bp repeats. Differences in VNTR lengthshave been found to impact drug metabolism in part by alter-ing gene expression. Metabolism of arsenic trioxide, an agentused in the treatment of promyelocytic leukemia, is dependenton the activity of arsenic methyltransferase (AS3MT). Short-ened lengths of VNTR sequences in the AS3MT 5′-untranslatedregion (UTR) has recently been found to be associated with in-creased AS3MT enzyme levels and activity (11). Analysis of therole of VNTRs in drug metabolism is a rapidly expanding area ofpharmacogenomics.

Drug metabolizing systems are generally well understood.However, testing for metabolic abnormalities is not routinelydone, in part due to the small population of affected individualsand the costs involved in esoteric testing. It is of significant ben-efit to the clinician who encounters these idiosyncratic reactionsto have a good understanding of their etiology and manage-ment in order to know when such testing is appropriate (phar-macogenetics). In addition, overexpression of drug targets andalterations in DNA repair systems also influence the antitumoractivity of drugs (pharmacogenomics). Gene variation and hap-lotypes associated with antineoplastic therapeutics such as Rit-uxan, thiopurines (TPMT), irinotecan and gemcitabine, amongothers, have been studied extensively (12–17). In this review,we evaluate the pharmacogenetics and pharmacogenomics of5FU/capecitabine, irinotecan, oxaliplatin, cetuximab, and beva-cizumab for CRC treatment, and aspirin and HMGCoA reduc-tase inhibitors (statins) for CRC prevention. This review willprovide an overview of current work in this area.

Table 1 lists the metabolizing enzymes related to the agentsused to treat colorectal cancer. The role of each enzyme systemwill be described more fully in an agent specific fashion.

FLUOROPYRIMIDINES: 5FU ANDCAPECITABINE

5FU is an analog of uracil that is converted intracellularlyinto 3 principle active metabolites: 5-fluoro-2′-deoxyuridine-5′-monophosphate (FdUMP), 5-fluoro-2′-deoxyuridine-5′-triphosphate (FdUTP), and 5-fluorouridine-5′-triphosphate

Pharmacogenomics of Colon Cancer 631

Dow

nloa

ded

By: [

CD

L Jo

urna

ls A

ccou

nt] A

t: 23

:23

20 M

ay 2

007

(FUTP). Both normal and tumor cells metabolize 5FU toFdUMP and FUTP, well known for their cytotoxic effects.FdUMP inhibits thymidylate synthase (TS), while FUTP canbe incorporated into RNA in place of uridine triphosphate,resulting in errors in RNA processing and protein synthesis.

The antitumor effects of 5FU are primarily mediated by itsmetabolite, FdUMP, which inhibits TS through the formation ofan extremely stable ternary complex composed of FdUMP, TS,and the cofactor 5,10-methylene-tetrahydrofolate (5,10-MTHF,CH2THF) (21). TS is an essential enzyme in DNA synthesis. Itcatalyzes the reductive methylation of dUMP in the final stepin the only de novo pathway for synthesis of dTMP. 5,10-CH2-H4-folate serves as a methyl donor and is converted into di-hydrofolate. Formation of the FdUMP-TS complex preventsmethylation of the deoxyuridine-5′-monophosphate (dUMP)into deoxythymidine-5′ -monophosphate (dTMP) catalyzed byTS, leading to depletion of thymidine and inhibition of DNAsynthesis.

TS and 5FU response

TS expression varies and is proportional to cellular sensitivityto 5FU, with increased TS levels associated with 5FU resistance(15–17). Several clinical trials have found a direct relationshipbetween increased intratumoral levels of TS and poorer out-comes (22–23). TS normally suppresses its own translation, andthis can be disrupted by FdUMP, leading to increased TS mRNAtranslation rates (24). Thus, 5FU can mediate upregulation ofTS levels, resulting in drug resistance. TS gene expression alsohas been related to polymorphisms of double-tandem (2R) ortriple-tandem (3R) 28 bp repeats in the promoter region of theTYMS gene (25). The homozygous triple tandem repeats geno-type (3R/3R) is associated with increased TS expression in vitroand higher TS activity in vivo compared to the double tandemrepeats genotype. Thus, patients with the triple tandem repeatsgenotype may have a reduced probability of deriving benefitfrom treatment with 5FU compared to the other genotype (22,23).

Recently, a single nucleotide polymorphism G>C has beendescribed at the twelfth nucleotide of the second repeat of the 3Rallele, leading to a tri-allelic locus (2R, 3RG, and 3RC) (26). Thispolymorphism changes a critical residue in the USF E-box con-sensus element. The 3RC allele showed transcriptional activitythat was similar to that of the 2R allele. Another common poly-morphism in the 3′-UTR of TYMS, a 6-bp deletion at bp 1494,recently has been characterized (27). A 4-fold decrease in TYMSexpression for patients homozygous for the deletion comparedwith patients who were homozygous for lack of the deletion(ins 6 bp/ins 6 bp) has been reported. Surprisingly, though, thehaplotype 2R/ins 6 bp was more significantly associated withincreased risk of 5FU related side effects rather than with treat-ment response (28). These authors also reported that the 2R/2Rhomozygotes were 20 times more likely to sustain Grade 3/4toxicity after treatment with 5FU. This was thought to occur be-cause lower levels of TS in normal tissues would promote DNAsynthesis inhibition and cell death after 5FU. These data sug-

gest that interactions between polymorphisms may be relevantto predicting drug response and toxicity.

At least 80 percent of 5FU is metabolized and inactivatedby dihydropyrimidine dehydrogenase (DPD) (29). The activityof this enzyme varies widely among human populations withup to 20-fold differences (30). Patients with low DPD activitylikely are to suffer greater risk of hematopoietic, neurologicaland gastrointestinal toxicity due to increased accumulation ofthe active metabolites of 5FU. These adverse effects can be fatalin some patients (31). Human DPD cDNA has been cloned andsequenced, and the genomic structure of the DPD gene has beenreported (29). DPD is encoded as a single copy gene (DPYD)of 23 exons on 1p22. The crystal structure of pig DPD has re-cently led to the elucidation of amino acids critical for enzymefunction. More than 40 mutations and polymorphisms have beenidentified, some of which result in decreased activity of the DPDenzyme (32). DPD gene (DPYD) polymorphism in humans oc-curs with an incidence of 3 percent who are heterozygous forDPD deficiency, and approximately one in 1,000 who are ho-mozygous (19). However, some investigators report that up to59 percent of patient’s may be affected by a DPD deficiency,with DPD enzyme activity comparable with that of obligate het-erozygotes (33). In homozygotes, total lack of activity of thisenzyme results in reduced pyrimidine metabolism, leading tothymidine-uraciluria and potential neurological disorders. Theheterozygote variant is less likely to manifest its phenotype inthe absence of 5FU challenge. A G->A transition at the 5′-splicesequence of exon 14 encompasses approximately 50 percent ofthe nonfunctional alleles. Processing of pre-mRNA bearing thismutation results in loss of exon 14 and the synthesis of a trun-cated protein. The resulting protein product is truncated by 55amino acids; its catalytic activity is virtually absent. While ithas been suggested to screen cancer patients routinely for theexon 14-skipping mutation before 5-FU treatment, there are atleast 17 variants of the allele that are associated with decreasedDPD activity. At this time, there are no definite data to reachconsensus on an optimal panel of genotypes that correlate withthe phenotype (2).

Capecitabine is a carbamate derivative of 5′-deoxy-5-fluorouridine, an oral 5FU prodrug better known as 5′-DFUR.Its activity depends on conversion to 5FU, making it subject tothe same pharmacogenetic parameters described for 5FU (34).It readily is absorbed in its prodrug form through the intes-tine, and is converted by carboxylesterases, in the liver, periph-eral tissues or in tumor to 5′-deoxy-5-fluorocytidine (5′-DFCR)(35). Cytidine deaminase, an enzyme found in most tissues, in-cluding tumors, subsequently converts 5′-DFCR to 5′-deoxy-5-fluorouridine (5′-DFUR). The enzyme thymidine phosphorylase(TP) then hydrolyzes 5′-DFUR to 5FU. Many tissues through-out the body express thymidine phosphorylase. Some humancarcinomas express this enzyme in higher concentrations thansurrounding normal tissues, resulting in tumor-selective gener-ation of 5FU (35). Thus, unlike 5FU, capecitabine may be moreactive in tumors expressing high levels of TP, although high tu-mor TS and DPD expression would still be expected to predictresistance to this agent (22). Clinical studies have demonstrated

632 H. Nguyen et al.

Dow

nloa

ded

By: [

CD

L Jo

urna

ls A

ccou

nt] A

t: 23

:23

20 M

ay 2

007

that for patients taking capecitabine, intratumor levels of 5FUmay be 3.5-fold higher than in normal tissues. This confers atheoretical advantage of capecitabine over 5FU in terms of ac-tive drug concentration in tumor tissues. Intestinal activation,on the other hand, may contribute to the occurrence of diarrhea(36). Several clinical studies have confirmed the general clinicalequivalence of infusional 5FU and oral capecitabine (37). Tumorlevels of TP can vary between patients, with low levels leading todiminished drug activation and drug resistance (19). TP plays anadditional role as platelet-derived endothelial cell growth factor,and its expression levels also are associated with tumor angio-genesis (20). This factor, therefore, may have implications fortreatment with bevaczumab, the monoclonal antibody directedagainst VEGF.

CAMPTOTHECINS: IRINOTECAN

Irinotecan is a prodrug that is converted in vivo to its ac-tive form SN-38 via the enzyme carboxylesterase (35, 38). SN-38 is 4–4,000 times more potent in inhibiting topoisomerase Icompared to CPT-11 and is responsible for the major toxicitiesincluding diarrhea and myelosuppression at high serum concen-trations. SN-38 is inactivated via glucuronide conjugation byUDP-glucuronosyl-transferase 1A1 (UGT1A1) and eliminatedvia bile and urine. Mutations in the coding region and differencesin the number of TA repeats of the promoter region of the genethat encodes for UGT1A1 are associated with reduced func-tion and/or expression of this enzyme (39–41). Reduced SN-38glucuronidation is associated with 7 as opposed to 6 TA re-peats. Twelve to 16 percent of patients express the homozygousgenotype TA7/TA7 (42). Patients who are either homozygous orheterozygous for the 7 TA repeats have a 7-fold greater likeli-hood of adverse effects including diarrhea and/or myelosuppres-sion with irinotecan exposure compared to those with wild-typegenotype of 6 TA repeats (41). UGT1A1 activity is reducedin individuals with polymorphisms of the UGT1A1*28 allele,which is homozygous in approximately 10 percent of the NorthAmerican population. Innocenti found that patients with the 7/7genotype (UGT1A1*28 homozygous) had a 9.3-fold greater riskof Grade 4 neutropenia compared with the patients with a 6/6 or6/7 genotype. Genotype testing of UGT1A1 may identify geno-type variants in approximately 50 percent of all cases of Grade4 neutropenia after a 300- to 350-mg/m2 infusion of irinote-can (41). Excluding patients with a UGT1A1*28 homozygousgenotype from receiving irinotecan could reduce the incidenceof Grade 4 neutropenia from 25 percent to 12 percent. Suchtesting also would mean that a significant proportion of eligiblepatients should avoid irinotecan therapy at the standard dosingschedule (43). The importance of these findings recently led theFDA to change the approved label describing the officially sanc-tioned use of CPT-11 in CRC treatment. On August 22, 2005,the FDA approved a molecular assay (Invader UGT1A1, ThirdWave Technologies, Inc., Madison, WI) for use in identifyingpatients that may be at increased risk of adverse reactions toirinotecan. The test detects and identifies specific mutations inUGT1A1. Clinical studies have shown the assay to be 100 per-cent accurate compared with DNA sequencing, the standard for

genotype determination (n = 285, 95 percent lower limit on con-fidence = 99 percent). According to recent updates in the safetylabeling for irinotecan, a one-level reduction in initial irinotecandose should be considered in patients known to be homozygousfor the UGT1A1*28 allele. The precise dose reduction requiredfor this patient population is unknown, suggesting that subse-quent dose modifications should be considered based on indi-vidual patient tolerance to treatment. This change constitutes thefirst use of pharmacogenetics to modify oncologic therapy.

Other polymorphisms in genes responsible for drugmetabolism may contribute to differential responses to irinote-can. Among these are polymorphisms in the genes encodingcarboxylesterase, and the polymorphism in exon 12 of the mul-tidrug resistance gene ABCB1 (C1236T), that result in increasedexposure to both irinotecan and its toxic metabolite SN-38(44, 45).

OXALIPLATIN

This third-generation platinum compound was approved bythe FDA for use in metastatic colorectal cancer in 2002 (46). Aswith other platinum compounds, oxaliplatin’s activity in colorec-tal cancer in large part is due to its ability to form intrastrand andinterstand DNA adducts eventually leading to apoptosis (47).The bulkier carrier group found on oxaliplatin versus cisplatin isthought to cause platinum-DNA adducts that are more cytotoxicthan those formed by other platinum agents such as cisplatinand carboplatin. Mismatch repair (MMR) deficiency, which iscommon in colorectal cancer, and an enhanced ability to by-pass the site of DNA damage have been demonstrated to causeresistance to cisplatin but not to oxaliplatin (47). Oxaliplatinresistance occurs through multiple mechanisms including de-creasing drug accumulation, enhanced tolerance, and increasedrepair of DNA adducts. High levels of DNA repair enzymes intumors confer resistance to platinum, while relatively low levelsof these enzymes generally have been associated with sensitivity.

The nucleotide excision repair cross-complementing (ERCC)gene family participates in the repair of platinum associatedDNA intrastrand crosslinks and may play a critical role in con-ferring resistance. This gene family is present in all mammaliancells and ERCC1 is a highly conserved enzyme that is specificto the nucleotide excision repair pathway and whose absence islethal. Shirota et al. found that the mRNA expressions of the en-zymes thymidine synthase (TS) and ERCC1 have a statisticallysignificant association with survival in metastatic colorectalcancer patients being treated with oxaliplatin-based chemother-apy (48). In addition to gene expression levels, polymorphismin ERCC1 also can have implications in treatment response (48,49). In addition, Park et al. also have described several clinicaloutcomes based on polymorphisms in the nucleotide excision re-pair xeroderma pignmentosum group D (XPD) gene, also knownas ERCC2. The median overall survival for patients with theglutamine/glutamine, lysine/glutamine and lysine/lysine pheno-types were 3.3, 12.8 and 17.4 months, respectively, for patientstreated with oxaliplatin and 5FU/leucovorin (50).

For enzymes in the base excision repair pathway,Stoehlmacher et al. found that polymorphisms in codon 399 of

Pharmacogenomics of Colon Cancer 633

Dow

nloa

ded

By: [

CD

L Jo

urna

ls A

ccou

nt] A

t: 23

:23

20 M

ay 2

007

the XRCC1 excision repair enzyme are associated with clinicaloutcome (49). It was noted by Stoehlmacher’s group that 73 per-cent of patients who responded to an oxaliplatin-based regimenwere found to carry the arginine/arginine genotype (51). How-ever, 66 percent of the patients who did not respond were foundto carry the glutamine/glutamine genotype. Patients with at leastone mutant allele had a 5.2-fold increase in the risk of failingoxaliplatin therapy.

Detoxification of oxaliplatin via efflux pumps beginswith conjugation of glutathione via various members of theglutathione-S transferase family- GSTP1, GSTM1, and GSTT1(52). GSTP1 commonly is overexpressed in malignant tumorsand may account in part for the inherent drug resistance in manymalignancies. Among patients treated with oxaliplatin-basedchemotherapy, overall survival was noted to be 24.9 months forpatients with the valine/valine homozygotes at position 105 ofthe GSTP1 gene. Isoleucine homozygotes had the worst survivalat 7.9 months and heterozygotes was intermediate at 13.3 months(52). Interestingly polymorphisms of GSTM1 and GSTT1 werenot found to be associated with clinical outcomes.

PREDICTIVE AND PROGNOSTIC PROFILES

A relevant role of pharmacogenomics is to assist in the pre-diction of drug response and patient prognosis. Recent studies onvarious biomarkers indicate that this area is maturing. Immunos-taining for TS, p53, and other proteins involved in cell replicationand apoptotic regulation has been studied as a potentially use-ful technique to identify patients more or less likely to benefitfrom treatment with 5FU or capecitabine (54). It is clear that thedeterminants of treatment response and outcomes will becomemore complex as we gain increased understanding of pathwaydisruption in cancer. One recent report included 23 indepen-dent biomarkers (pRb, p16, p21, p27, p53, proliferating cell nu-clear antigen, cyclin D1, bcl-2, epidermal growth factor receptor,C-erb-B2, topoisomerase-I, liver fatty acid-binding protein, ma-trix metalloproteinases (MMP) 1-3, 7, 9, and 13, MT1-MMP,MT2-MMP, and tissue inhibitors of MMP 1-3) (55). Using mul-tivariate analysis of these markers evaluated on paraffin embed-ded tumor sections from 90 patients with Stage III colorectalcancer, the complete marker profile was independently the mostsignificant prognostic factor (hazard ratio, 2.27; 95% CI, 1.15–4.48; P = 0.004).

Gene expression profiling also has been carried out to de-termine if specific transcript patterns are linked to clinical out-omes. Barrier et al. assessed microarray gene expression to builda prognosis predictor for Stage II and III colon cancer patients.Tumor and nonneoplastic mucosa mRNA samples from 18 pa-tients (nine with a recurrence, nine with no recurrence) wereprofiled using the Affymetrix HGU133A GeneChip. A 30-genetumor tissue-predictor and a 70-gene nonneoplastic mucosa-based predictor were developed with estimated accuracies topredict recurrence of 78 and 83 percent, respectively (56). An-other group who investigated the use of Affymetrix arrays todefine prognosis for Stage II colon cancer evaluated RNA sam-ples from 74 patients. A 23-gene signature was identified that

predicted recurrence (57). They validated their training set in 36independent patients. The overall performance accuracy was 78percent.

A number of biomarkers of drug response and metabolismhave been described above, while only a handful are availablefor routine use. Genomic profiling is still not a practical clinicaltool, and the pathologist remains as the principle guardian oftumor material. In order for bioprofiling to penetrate into rou-tine use, prospective clinical trials need to be performed thatcan validate the relevance and utility of bioprofiling and phar-macogenomic testing. Such testing will need to be reproducibleand cost effective.

TARGETED AGENTS

Cetuximab

Cetuximab is a monoclonal antibody that recently wasapproved for use in colorectal adenocarcinoma as eithera monotherapy or combination therapy with irinotecan inirinotecan-refractory patients. Cetuximab has a high affinityand specificity for epidermal growth factor receptor (EGFR),a member of the ErbB family of receptors. EGFR has become ahigh-profile target in colorectal cancer due to the association ofcolorectal cancer with EGFR overexpression or mutation (58).In its active form, EGFR complexes with epidermal growthfactor (EGF), leading to dimerization with other ErbB recep-tors, subsequently triggering an intracellular cascade of events.Cetuximab acts by complexing with cell surface EGF recep-tors, preventing EGF-induced activation of EGFR and inducingreceptor internalization (59). Currently, EGFR expression hasbeen only sporadically associated with treatment response (60).Skin toxicity (an acne-like rash) is also weakly associated withtreatment response (61).

Polymorphisms in the EGFR gene were postulated to be pre-dictive of clinical response in metastatic colorectal cancer pa-tients (62, 63). EGFR overexpression may result from a highlevel of EGFR translation. Gebhardt et al. observed that tran-scription of EGFR gene inversely was related to the length ofdinucleotide repeats (CA repeats) in a highly polymorphic re-gion located in intron 1; thus, patients with less repeats wereassociated with increased transcription of EGFR (62). Resultsfrom a preliminary retrospective study showed that metastaticcolorectal cancer patients with longer CA repeats demonstrated asuperior clinical response to oxaliplatin therapy (63). Among thepatients assessed by Zhang et al., those possessing <20 EGFRCA repeats were more likely to show disease progression thanwere patients with ≥20 CA repeats (P = 0.019; log-rank test)(63). IL-8 was also noted by Zhang et al. to play a downstreamrole in EGFR signaling. Patients with the IL-8 receptor CXCR1GC genotype were found to have an increased relative risk oftime to tumor progression that was 1.55 (95% CI, 0.8–3.0) timesthat of patients with the homozygous GG genotype (P = 0.17;log-rank test). Their data suggest that gene polymorphisms ac-tive in the EGFR pathway may be associated with the sensitivityof colorectal cancer patients to platinum-based chemotherapy.

634 H. Nguyen et al.

Dow

nloa

ded

By: [

CD

L Jo

urna

ls A

ccou

nt] A

t: 23

:23

20 M

ay 2

007

Bevacizumab

Bevacizumab is a monoclonal antibody recently approved foruse in combination with 5-fluorouracil-based chemotherapy fortreatment of patients with metastatic colorectal carcinoma. It is ahumanized monoclonal antibody that prevents vascular endothe-lial growth factor-A (VEGF-A) from binding to its receptorsVEGFR-1 and VEGFR-2. VEGF overexpression is associatedwith tumor angiogenesis in multiple types of tumors. Tumorcells induce hypoxia through the hypoxia inducible factor-1α

(HIF-1 α) pathway. Induction of this pathway leads to transcrip-tion of various factors such as VEGF-A. Uninhibited, VEGF-Abinds to its receptors located on the surface of endothelial cellsresulting in increased blood vessel permeability, angiogenesisand the proliferation of endothelial cells.

Levels of VEGF have been investigated in multiple tumortypes such as colorectal and breast. Over 30 SNPs have beenidentified in the VEGF gene (64). However, the relationshipbetween measured VEGF levels and response to bevacizumabremains to be defined (65). Karp studied serum VEGF levelsin adult AML patients receiving bevacizumab, and monitoredchanges in levels after treatment. Of the 15 patients studied, 10had complete suppression of VEGF 2 hours post bevacizumabinfusion, while 4 cases were found to have only 24 percent to72 percent suppression. These data suggest that pharmacoki-netic differences in VEGF levels after treatment may contributeto variations in treatment response. Circulating VEGF levelstend to rise after bevacizumab therapy, and the degree of in-crease varies among patients (66). Thus, posttreatment fluctua-tions in VEGF levels may also influence treatment response.In addition to colorectal cancer, bevacizumab therapy is be-ing investigated for pancreatic, prostate, renal, hepatic cancer,melanoma, and leukemia. In light of the possibility of its var-ied use, any information guiding appropriate dosing of beva-cizumab to increase efficacy or reduce toxicity would be of greatimportance.

Chemoprevention

Many of the gene mutations that contribute to highly pene-trant cancer risk have been discovered using an approach thattakes advantage of highly informative families with many af-fected relatives and a well characterized phenotype. Wholegenome linkage analysis has been the most successful strat-egy to identify rare, highly penetrant gene mutations relevant tocancer. Their identification has allowed intensive early detectionsurveillance strategies to prevent cancer and improve survival(67). Newer strategies such as sib-pair studies have recentlyidentified candidate regions of interest and are likely to yieldcontributing genes as well, but these approaches often have dif-ficulties mapping genetic intervals down to the resolution nec-essary for disease gene identification. Genotyping technologyrecently has evolved to increase the resolution of genome scansby several orders of magnitude. Now, association studies canmeasure how genetic variation contributes to cause specific in-dividuals to be at high risk of malignancy. In the near future stud-ies extending beyond the characterization of rare highly pene-

trant genetic syndromes should clarify the relationship betweenindividual genomic variation and clinical response to specificcancer chemoprevention and therapeutic agents for colorectalcancer.

Cycloxygenase-2 (COX-2) inhibitors

Cancer chemoprevention has identified specific pharmaco-logic and/or dietary agents that decrease incidence of specificcancers. Perhaps the best known examples are that of aspirinand other non-steroidal anti-inflammatory agents (NSAIDs) incolorectal cancer (CRC) prevention. In terms of CRC, this areahas been characterized extensively in the high-penetrance ge-netic CRC syndromes. Because the progression from initiationof adenoma to carcinoma can take several years, prevention canhave a major impact on morbidity and mortality. NSAIDs andthe more selective cycloxygenase-2 (COX-2) inhibitors havedemonstrated significant chemopreventative benefits in Famil-ial Adenomatous Polyposis (FAP). Four multi-arm random-ized, placebo controlled trials have demonstrated that sulindac300–400 mg/day and celecoxib 800 mg/day significantly reduceboth the number and size of rectal adenomas in FAP patients (forrecent review, see Keller, 2003) (68). When combined, these datasuggested that treatment (21 subjects total) for 4–6 months withsulindac at 300–400 mg/day resulted in a ∼70 percent decreasein the rectal adenoma rate, while patients treated with Celecoxib800 mg po qD for 6 months (27 patients total) experienceda ∼25 percent drop. Currently, ongoing chemoprevention tri-als for HNPCC include a randomized, placebo-controlled PhaseI/II multicenter trial evaluating the safety and efficacy of cele-coxib in ∼80 HNPCC subjects (69). This study compares 3 pro-posed interventions: celecoxib at 200 mg po BID for 12 months,400 mg po BID for 12 months, and placebo po BID for12 months. The effects of these treatment arms on a number ofendoscopic and tissue-based biomarker endpoints will be eval-uated at baseline and 12 months. The Concerted Action PolypPrevention (CAPP2) study evaluates the preventative effects ofaspirin and resistant starch in HNPCC carriers. Patients are ran-domized to receive 600 mg enteric coated aspirin or placeboand 30 g treatment starch or placebo. The primary endpoint ofthe study will be the number, size, and histological stage ofCRC found after 2 years on treatment or placebo. In summary,studies demonstrate the benefit of chemoprevention in a genet-ically defined group of patients with highly penetrant diseasesusceptibility.

Statins in chemoprevention

Recently, it has been discovered that HMG CoA-Reductaseinhibitors (“statins” such as atovarstatin, pravachol or simvas-tatin) primarily used to prevent cardiovascular disease have sig-nificant chemoprevention activity in multiple cancers, but mostimpressively in colorectal cancer (70, 71). While the statinshave been studied extensively in clinical trials assessing car-diovascular risk, most studies have not had either long-termfollow up or the appropriate institutional approvals to link toCRC related outcomes. However, this endpoint was assessed

Pharmacogenomics of Colon Cancer 635

Dow

nloa

ded

By: [

CD

L Jo

urna

ls A

ccou

nt] A

t: 23

:23

20 M

ay 2

007

in the Cholesterol and Recurrent Events (CARE) trial, whichwas designed primarily to evaluate the long-term effects ofpravastatin on plasma concentration of C-reactive protein. Asignificant reduction in CRC risk was found in pravastatin users(12 CRCs) vs the placebo group (21 CRC, O.R. = 0.57). Thisobservation has become more intriguing with the subsequent as-sociation of elevated CRP levels with elevated CRC risk (69).The relatively small number of CRC events in the CARE trialhas made drawing conclusions about this relationship difficult.Graaf et al. recently conducted a large-scale retrospective studyof statin use and risk of all types of cancer. Using the PHARMOdatabase from the Netherlands, they identified more than 3,000statin users from pharmacy records and almost 17,000 matchedcontrols. Their data suggested that statins are protective whenused longer than 4 years (adjusted OR, 0.64; 95% CI), althoughthere was no breakdown available by individual types of can-cer (70). Recently, robust new data were presented supporting arole for statin drugs in CRC prevention (71). The hypothesis thatstatin use is associated with CRC prevention was tested directlyin this large study (1814 CRC cases and 1959 control subjects) ofthe Israeli population-based Molecular Epidemiology of ColonCancer cohort (58, 60). Medication history was confirmed bypersonal interviews, and 96.5 percent of reported statin use wasconfirmed by prescription records with the Israeli Health Ser-vice records. Odds ratios were used to estimate relative risk andlogistic regression used to adjust for other risk factors. Compar-ing subjects using statins for >5 years to nonusers, overall a 51percent relative risk reduction was observed for CRC (OR = 0.4995% CI, 0.38– 0.62; p < 0.0001) (71). When adjusted for possi-ble confounding factors between case and control subject groupsof aspirin/NSAID use, ethnicity, family history of CRC, physicalactivity, dietary vegetable intake, and hypercholesterolemia, theadjusted OR was 0.54 (95% CI, 0.39–0.75; p < 0.0001). Sim-ilar results were seen when subsets were separately analyzedfor colon (OR = 0.53 95% CI, 0.36–0.76 p < 0.0007) and rectal(OR = 0.38 95% CI, 0.19–0.73; p < 0.0038) cancers, suggest-ing the effects of statins are not GI-tract location-specific andshare mechanisms common to both anatomical sites. Similarrisk reduction profiles were observed when analyzed separatelyfor Pravastatin (OR = 0.45 95% CI, 0.31–0.64; p < 0.0001)or Simvastatin (OR = 0.47 95% CI, 0.34–0.65; p < 0.0001)users (OR = 1.0 95% CI, 0.55–1.93; p = 0.936, suggesting thechemopreventive effects are not “brand specific.” No evidenceof synergy between statins and aspirin/NSAIDs was observed(p = 0.54).

Chemoprevention of sporadiccolorectal cancer

The contribution of human genetic variation to chemopreven-tion of sporadic cancer is poorly defined at this point in time.It is well appreciated that human pharmacologic response toNSAID treatment is highly variable. In terms of cardiovascu-lar risk prevention, it is striking than 25% of individuals takingaspirin do not respond by increased bleeding time or other sig-nificant aspects of platelet dysfunction in clotting (71). Like

aspirin, in clinical users of HMG coA reductase inhibitor/statindrugs for cardiovascular risk reduction, there is wide variationin inter-individual response to statin therapy. It has been hy-pothesized that genetic differences significantly contribute tothis variation (62–66). Recently, with reference to the use of ge-netic screening to guide lipid-lowering therapy, genetic analysesof 1,536 subjects from the PRINCE cardiovascular risk reduc-tion clinical trial were used to test the hypothesis that commongenetic variants influence the degree of lipid level reductionduring pravastatin therapy (72). Candidate cholesterol synthesisand transport genes were selected for study, and genetic varia-tion was examined for associations with changes in lipid levelsduring pravastatin therapy. Two tightly linked SNPs occurring ina haplotype from the HMGCR gene were found to be associatedwith a 22% reduction in pravastatin therapy lipid lowering effi-cacy (P < 0.001) (62). Interestingly, the SNPs in this haplotypedid not affect lipid levels in a second group of 649 subjects whodid not receive pravastatin therapy (72). Nonstatistically signif-icant trends also were found for SNPs in the squalene synthaseand cholesteryl ester transfer protein genes (72). The investi-gators concluded that the cholesterol synthesis genetic variantsidentified in their study are likely to contribute significantly toimpaired lipid lowering responses in treated subjects.

CONCLUSION

Current paradigms in chemotherapy do not effectively ac-count for interpatient variability in the expression of relevanttarget enzymes. This leads to unpredictable disease responseand patient toxicities. With advancing technology, it is expectedthat the information gleaned from pharmacogenetics will even-tually be used to individualize treatment, thereby maximizingeffects and minimizing toxicities of chemotherapy. Recent ap-proval by the FDA of the first gene chip for human testing, theAmpliChip Cytochrome P450 Genotyping Test, that analyzesone of the p450 family members, is a step in the right direc-tion. As new diagnostic platforms develop, it is anticipated thatfuture generations of physicians will be able to profile their pa-tients “metabolomic” profile, making it possible to tailor drugsand their dosages appropriately. Current commercially availableimmunohistochemical profiling of tumor tissue to assay drug tar-gets, such as TS, have made a limited impact on practice patternsin part due to a limited number of drugs available to treat CRC.However, with an increased number of new drugs and clinicaltrials linking outcomes with biomarker profiling, the next decadeshould see a dramatic increase in the utility of pharmacogeneticprofiling for treatment selection. Can SNPs and haplotypes beapplied to chemoprevention and therapy? The authors are hope-ful that we will see significant progress in the coming years. Theability to identify genetic markers associated with cancer pre-vention has greatly advanced in recent years. High-throughputtechnologies have also evolved, and now allow economical andrapid large-scale genotyping of large patient groups in thera-peutic clinical trials. Sets of SNPs can be genotyped across agenomic segment to characterize the specific chromosomes thatexist in a population; the combination of alleles that exist along a

636 H. Nguyen et al.

Dow

nloa

ded

By: [

CD

L Jo

urna

ls A

ccou

nt] A

t: 23

:23

20 M

ay 2

007

chromosome is termed a haplotype. Haplotypes are a combina-tion of alleles at different markers along the same chromosomethat are inherited as a unit (62, 64). The fundamental differ-ence between haplotypes and individual genotypes at SNPs isthat the alleles are assigned to a chromosome. In essence, eachindividual has 2 haplotypes for a given stretch of the genome,representing the maternal and paternal chromosomes. Haplo-types provide increased statistical power to detect associationand map etiological mutations over classical single-marker ap-proaches, as well as greatly reducing the amount of genotypingrequired to evaluate the role of common genetic variation ona clinical outcome. The HapMap and Perlegen initiatives haveidentified sets of SNPs that will characterize the common haplo-types across the human genome in the major ethnic groups. Thisinitiative, combined with other public efforts to identify SNPsacross specific gene regions, will greatly reduce the amount ofgenotyping required for genome-wide or candidate gene associ-ation studies of clinical phenotypes including cancer preventionand drug response.

The technology and bioinformatic resources for first gener-ation Whole Genome Association (WGA) studies to identifyphenotypically important genes related to clinical responseto specific chemoprevention agents for cancer have arrived.The technical aspects of genotyping are now in place, andstatistical methods have been developed and implemented inother studies. Recently a large-scale, case-control study suc-cessfully identified at least one susceptibility gene to a complexgenetic trait, yet this study of myocardial infarction providesan important background for the whole genome study ofchemoprevention responses. In a study of 1,133 cases and 1,006controls, Ozaki et al. identified a gene on 6p21 associated withsusceptibility to myocardial infarction (77). The authors studied92,788 SNPs, and 65,671 (70.8 percent) of these SNPs could begenotyped successfully. Using a high-throughput genotypingstrategy permitted them to screen 13,738 genes. One of thesegenes, lymphotoxin-α (LTA), was associated with an increasedrisk of myocardial infarction, OR = 1.78, p = 0.00000033.This association was validated in a replication dataset withcareful control for population stratification. Functional studiesidentified 2 SNPs with recognizable mechanisms, one of whichinduces a cell adhesion molecule expressed in vascular smoothmuscle cells of human coronary arteries, and the other that leadsto increased expression of LTA. Although whole genome associ-ation studies for cancer prevention have not been completed yet(as far as we are aware), the technical aspects of genotyping arenow in place, the statistical methods have been developed andimplemented in other settings. Existing specimens from clinicaltrial resources are well-positioned to take advantage of this ap-proach to better understand genetic contributions to both cancerprevention and therapeutic response. There are currently at least6 ongoing WGA studies: 1) Whole Genome Association Studyof HDL Level Modifiers (PI Kelly Frazer, Pfizer, New York);2) Colorectal Cancer (PI Stephen B. Gruber, University ofMichigan, Ann Arbor, MI); 3) Autism (PI Aravinda Chakravarti,Johns Hopkins University, Baltimore, MD); 4) Alzheimer Dis-ease (PI Jeff Trent, Translational Genomics Institute, Phoenix,

AZ); 5) Swedish and American Type II Diabetes Determinants(PI David Altschuler, Broad Institute, Cambridge MA); and6) Whole Genome Association Scan for Type II Diabetes inthe Finnish Population (PI Francis Collins, NHGRI, Bethesda,MD). Three of these studies (1,2 and 6) utilize Perlegen WGAscanning technology, 2 (3, and 5) use Illumina BeadArray Tech-nology, and one (4) uses Affymtrix High Density 500 K SNPMapping Chips. (N.B.: The specific pros and cons of each tech-nology are beyond the scope of the discussion for this review).

In summary, it is clear that human pharmacologic responseto chemoprevention and chemotherapeutic agents for CRC ishighly variable, and that much of this variation is attributableto genetic factors (78). A new generation of mapping tools andtechnologies has been developed that exploit recent advancesin our knowledge of the roles that SNPs and haplotypes. Theseadvances allow an unprecedented level of precision to map thecontribution of genetic factors to cancer chemoprevention. Us-ing the examples of NSAID and statin chemoprevention of CRCas a model, there are both ongoing and completed large scaleclinical trials in which it is anticipated that genetic stratificationcan help identify patient subpopulations demonstrating clinicalbenefits that might otherwise be diluted and not recognized in agroup of heterogeneous admixed cancer subtypes with differentbiological causes. The inclusion of genetic stratification is likelyto help identify a high-risk patient population at increased cancerrisk who are especially likely to benefit from statin or NSAIDchemoprevention. Furthermore, because of recent requests bythe U.S. Food and Drug Administration to accept pharmacoge-netic data in new drug applications, SNPs and haplotypes pro-vide the underlying biological rationale for genetically stratifiedsecondary endpoint analyses of chemoprevention trials that failto demonstrate statistically significant benefit in the entire intentto treat trial population. While this review has focused on phar-macogenetics, many nongenetic factors contribute to differencesin treatment response and the toxic effects of chemotherapeuticagents. There is no substitute for close observation of the patientat the bedside.

REFERENCES1. Rougier, P.; Lepere, C. Metastatic colorectal cancer: first- and

second-line treatment in 2005. Semin Oncol. 2005, 32 (6 Suppl.8),15–20.

2. Petros, W.P.; Evans, W.E. Pharmacogenomics in cancer therapy: ishost genome variability important? Trends Pharmacol. Sci. 2004,25, 457–464.

3. Eichelbaum, M.; Ingelman-Sundberg, M.; Evans, W.E. Pharma-cogenomics and Individualized Drug Therapy. Annu. Rev. Med.2005. (epub ahead of print)

4. Evans, W.E.; McLeod, H.L. Pharmacogenomics–drug disposition,drug targets, and side effects. N. Engl. J. Med. 2003, 348, 538–549.

5. Hinds, D.A.; et al. Whole-genome patterns of common DNA varia-tion in three human populations. Science 2005, 307, 1072–1079.

6. Collins, F.S. Genome research: the next generation. Cold SpringHarb. Symp. Quant. Biol. 2003, 68, 49–54.

7. Crawford, D.C.; Akey, D.T.; Nickerson, D.A. The patterns of natu-ral variation in human genes. Annu. Rev. Genomics Hum. Genet.2005, 6, 287–312.

Pharmacogenomics of Colon Cancer 637

Dow

nloa

ded

By: [

CD

L Jo

urna

ls A

ccou

nt] A

t: 23

:23

20 M

ay 2

007

8. Crawford, D.C.; Nickerson, D.A. Definition and clinical importanceof haplotypes. Annu. Rev. Med. 2005, 56, 303–320.

9. Coulthard, S.; Hogarth, L. The thiopurines: an update. Invest. NewDrugs. 2005, 23, 523–532.

10. Wang, L.; Nguyen, T.V.; McLaughlin, R.W.; Sikkink, L.A,;Ramirez-Alvarado, M.; Weinshilboum, R.M. Human thiopurineS-methyltransferase pharmacogenetics: variant allozyme misfold-ing and aggresome formation. Proc. Natl. Acad. Sci. USA 2005,102, 9394–9399.

11. Wood, T.C.; Salavaggione, O.E.; Mukherjee, B.; Wang, L.; Klumpp,A.F.; et al. Human arsenic methyltransferase (AS3MT) pharmaco-genetics: Gene resequencing and functional genomics studies. J.Biol. Chem. 2006. (epub ahead of print)

12. Weng, W.K.; Levy, R. Two immunoglobulin G fragment C recep-tor polymorphisms independently predict response to rituximab inpatients with follicular lymphoma. J. Clin. Oncol. 2003, 21, 3940–3947.

13. Evans, W.E. Pharmacogenetics of thiopurine S-methyltransferaseand thiopurine therapy. Ther. Drug Monit. 2004, 26, 186–191.

14. Marsh, S.; McLeod, H.L. Pharmacogenetics of irinotecan toxicity.Pharmacogenomics 2004, 5, 835–843.

15. Fukunaga, A.K.; Marsh, S.; Murry, D.J.; Hurley, T.D.; McLeod, H.L.Identification and analysis of single-nucleotide polymorphisms inthe gemcitabine pharmacologic pathway. Pharmacogenomics J.2004, 4, 307–314.

16. McLeod, H.L.; King, C.R.; Marsh, S. Application of pharmacoge-nomics in the individualization of chemotherapy for gastrointestinalmalignancies. Clin. Colorectal. Cancer. 2004, 4, S43–47.

17. Marsh, S.; McLeod, H.L. Cancer pharmacogenetics. Br. J. Cancer2004, 90, 8–11.

18. Mandola, M.V.; Stoehlmacher, J.; Muller-Weeks, S.; et al. A novelsingle nucleotide polymorphism within the 5′ tandem repeat poly-morphism of the thymidylate synthase gene abolishes USF-1 bind-ing and alters transcriptional activity. Cancer Res. 2003, 63, 2898–2904.

19. Jakob, C.; Aust, D.E.; Meyer, W.; Baretton, G.B.; Schwabe, W.; et al.Thymidylate synthase, thymidine phosphorylase, dihydropyrimi-dine dehydrogenase expression, and histological tumour regres-sion after 5-FU-based neo-adjuvant chemoradiotherapy in rectalcancer. J. Pathol. 2004, 204, 562–568.

20. Matsuura, T.; Kuratate, I.; Teramachi, K.; Osaki, M.; Fukuda, Y.; Ito,H. Thymidine phosphorylase expression is associated with bothincrease of intratumoral microvessels and decrease of apoptosisin human colorectal carcinomas. Cancer Res. 1999, 59, 5037–5040.

21. Carreras, C.W.; Santi, D.V. The catalytic mechanism and structureof thymidylate synthase. Annu Rev Biochem 1995, 64, 721–762.

22. Edler, D.; Gilmelius, B.; Hallstrom, M.; Jakobsen, A.; Johnston,P.G.; Magnusson, I.; et al. Thymidylate synthase expression in col-orectal cancer: a prognostic and predictive marker of benefit fromadjuvant fluorouracil-based chemotherapy. J. Clin. Oncol. 2002,20, 1721–1728.

23. Allen, W.L.; Johnston, P.G. Role of genomic markers in colorectalcancer treatment. J. Clin. Oncol. 2005, 23, 4545–4552.

24. Tai, N.; Schmitz, J.C.; Liu, J.; Lin, X.; Bailly, M.; et al. Translationalautoregulation of thymidylate synthase and dihydrofolate reduc-tase. Front. Biosci. 2004, 9, 2521–2526.

25. Villafranca, E.; Okruzhnov, Y.; Dominguez, M.A.; Garcia-Foncillas,J.; Azinovic, I.; et al. Polymorphisms of the repeated sequencesin the enhancer region of the thymidylate synthase gene promotermay predict downstaging after preoperative chemoradiation in rec-tal cancer. J. Clin. Oncol. 2001, 19, 1779–1786.

26. Mandola, M.V.; Stoehlmacher, J.; Muller-Weeks, S. et al. A novelsingle nucleotide polymorphism within the 5′ tandem repeat poly-morphism of the thymidylate synthase gene abolishes USF-1 bind-ing and alters transcriptional activity. Cancer Res. 2003, 63, 2898–2904.

27. Ulrich, C.M.; Bigler, J.; Velicer, C.M.; Greene, E.A.; Farin, F.M.;Potter, J.D. Searching expressed sequence tag databases: discov-ery and confirmation of a common polymorphism in the thymidy-late synthase gene. Cancer Epidemiol. Biomark. Prev. 2000, 9,1381–1385.

28. Lecomte, T.; Ferraz, J.M.; Zinzindohoue, F.; Loriot, M.A.; Tregouet,D.A.; et al. Thymidylate synthase gene polymorphism predicts tox-icity in colorectal cancer patients receiving 5-fluorouracil-basedchemotherapy. Clin. Cancer Res. 2004, 10, 5880–5888.

29. Ridge, S.A.; Sludden, J.; Brown, O.; Robertson, L.; Wei, X.;et al. Dihydropyrimidine dehydrogenase pharmacogenetics inCaucasian subjects. Br. J. Clin. Pharmacol. 1998, 46, 151–156.

30. McLeod, H.L.; Sludden. J.; Murray, G.I.; Keenan, R.A.; David-son, A.I.; et al. Characterization of dihydropyrimidine dehydroge-nase in human colorectal tumours. Br. J. Cancer 1998, 77, 461–465.

31. van Kuilenburg, A.B.; Muller, E.W.; Haasjes, J.; Meinsma, R.;Zoetekouw, L.; et al. Lethal outcome of a patient with a completedihydropyrimidine dehydrogenase (DPD) deficiency after adminis-tration of 5-fluorouracil: frequency of the common IVS14 + 1G > Amutation causing DPD deficiency. Clin. Cancer Res. 2001, 7,1149–1153.

32. Van Kuilenburg, A.B.; Meinsma, R.; Beke, E.; Bobba, B.; Boffi, P.;et al. Identification of three novel mutations in the dihydropyrimidinedehydrogenase gene associated with altered pre-mRNA splicingor protein function. Biol. Chem. 2005, 386, 319–324.

33. Van Kuilenburg, A.B.; Haasjes, J.; Richel, D.J.; Zoetekouw, L.; VanLenthe, H.; et al. Clinical implications of dihydropyrimidine dehy-drogenase (DPD) deficiency in patients with severe 5-fluorouracil-associated toxicity: identification of new mutations in the DPDgene. Clin. Cancer Res. 2000, 6, 4705–4712.

34. Park, D.J.; Stoehlmacher, J.; Zhang, W.; Tsao-Weim D.; Groshen,S.; Lenz, H.J. Thymidylate synthase gene polymorphism pre-dict response to capecitabine in advanced colorectal cancer. Int.J. Colorectal Dis. 2002, 17, 46–49.

35. Rooseboom, M.; Commander, J.N.; Vermeulen, N.P. Enzyme-catalyzed activation of anticancer prodrugs. Pharmacol. Rev. 2004,56, 53–102.

36. Van Cutsem, E.; Twelves, C.; Cassidy, J.; et al. Oral capecitabinecompared with intravenous fluorouracil plus leucovorin in patientswith metastatic colorectal cancer: results of a large phase III study.J. Clin. Oncol. 2001, 19, 4097–4106.

37. Twelves, C.; Wong, A.; Nowacki, M.P.; et al. Capecitabine as ad-juvant treatment for stage III colon cancer. N. Engl. J. Med. 2005,352, 2696–2704.

38. Humerickhouse, R.; Lohrbach, K.; Li, L.; Bosron, W.F.; Dolan,M.E. Characterization of CPT-11 hydrolysis by human liver car-boxylesterase isoforms hCE-1 and hCE-2. Cancer Res. 2000, 60,1189–1192.

39. Kitagawa, C.; Ando, M.; Ando, Y.; Sekido, Y.; Wakai, K.; et al.Genetic polymorphism in the phenobarbital-responsive enhancermodule of the UDP-glucuronosyltransferase 1A1 gene and irinote-can toxicity. Pharmacogenet Genomics. 2005, 15, 35–41.

40. Iyer, L.; Hall, D.; Das, S.; Mortell, M.A.; Ramirez. J.; et al.Phenotype-genotype correlation of in vitro SN-38 (active metabo-lite of irinotecan) and bilirubin glucuronidation in human liver tis-sue with UGT1A1 promoter polymorphism. Clin. Pharmacol. Ther.1999, 65, 576–578.

41. Innocenti, F.; Undevia, S.D.; Iyer, L.; Chen, P.X.; Das, S.; et al.Genetic variants in the UDP-glucuronosyltransferase 1A1 genepredict the risk of severe neutropenia of irinotecan. J. Clin. Oncol.2004, 22, 1382–1388.

42. Rouits, E.; Boisdron-Celle, M.; Dumont, A.; Guerin, O.; Morel, A.;Gamelin, E. Relevance of different UGT1A1 polymorphisms inirinotecan-induced toxicity: a molecular and clinical study of 75patients. Clin. Cancer Res. 2004, 10, 5151–5159.

638 H. Nguyen et al.

Dow

nloa

ded

By: [

CD

L Jo

urna

ls A

ccou

nt] A

t: 23

:23

20 M

ay 2

007

43. McLeod, H.L.; Watters, J.W. Irinotecan pharmacogenetics: is ittime to intervene? J. Clin. Oncol. 2004, 22, 1356–1359.

44. Khanna, R.; Morton, C.L.; Danks, M.K.; Potter, P.M. Proficientmetabolism of irinotecan by a human intestinal carboxylesterase.Cancer Res. 2000, 60, 4725–4728.

45. Sai, K.; Kaniwa, N.; Itoda, M.; Saito, Y.; Hasegawa, R.; et al. Hap-lotype analysis of ABCB1/MDR1 blocks in a Japanese populationreveals genotype-dependent renal clearance of irinotecan. Phar-macogenetics 2003, 13, 741–757.

46. Raymond, E.; Faivre, S.; Woynarowski, J.M.; Chaney, S.G. Ox-aliplatin: mechanism of action and antineoplastic activity. Semin.Oncol. 1998, 25, 4–12.

47. Chaney, S.G.; Campbell, S.L.; Bassett, E.; Wu, Y. Recognition andprocessing of cisplatin- and oxaliplatin-DNA adducts. Crit. Rev.Oncol. Hematol. 2005, 53, 3–11.

48. Shirota, Y.; Stoehlmacher, J.; Brabender, J.; Xiong, Y.P.; Uetake,H.; et al. ERCC1 and thymidylate synthase mRNA levels predictsurvival for colorectal cancer patients receiving combination ox-aliplatin and fluorouracil chemotherapy. J. Clin. Onco.l 2001, 19,4298–4304.

49. Stoehlmacher, J.; Park, D.J.; Zhang, W.; Yang, D.; Groshen, S.;et al. A multivariate analysis of genomic polymorphisms: predic-tion of clinical outcome to 5-FU/oxaliplatin combination chemother-apy in refractory colorectal cancer. Br. J. Cancer 2004, 91, 344–354.

50. Park, D.J.; Stoehlmacher, J.; Zhang, W.; Tsao-Wei, D.; Groshen,S.; Lenz, H.J. A Xeroderma pigmentosum group D gene polymor-phism predicts clinical outcome to platinum-based chemotherapyin patients with advanced colorectal cancer. Cancer Res. 2001,61, 8654–8658.

51. Stoehlmacher, J.; Ghaderi, V.; Iobal, S.; Groshen, S.; Tsao-Wei, D.;Park, D.; Lenz, H.J. A polymorphism of the XRCC1 gene predictsfor response to platinum based treatment in advanced colorectalcancer. Anticancer Res. 2001, 21, 3075–3079.

52. Stoehlmacher, J.; Park, D.J.; Zhang, W.; Groshen, S.; Tsao-Wei,D.D.; et al. Association between glutathione S-transferase P1,T1, and M1 genetic polymorphism and survival of patients withmetastatic colorectal cancer. J. Natl. Cancer Inst. 2002, 94, 936–942.

53. Spano, J.P.; Lagorce, C.; Atlan, D.; Milano, G.; Domont, J.; et al.Impact of EGFR expression on colorectal cancer patient prognosisand survival. Ann Oncol 2005, 16, 102–108.

54. Watson, N.F.; Madjd, Z.; Scrimegour, D.; Spendlove, I.; Ellis, I.O.;et al. Evidence that the p53 negative/Bcl-2 positive phenotype isan independent indicator of good prognosis in colorectal cancer:a tissue microarray study of 460 patients. World J. Surg. Oncol.2005, 3, 47–57.

55. Lyall, M.S.; Dundas, S.R.; Curran, S.; Murray, G.I. Profiling mark-ers of prognosis in colorectal cancer. Clin. Cancer Res. 2006, 12,1184–1191.

56. Barrier, A.; Lemoine, A.; Boelle, P.Y.; Tse, C.; Brault, D.; et al.Colon cancer prognosis prediction by gene expression profiling.Oncogene 2005, 24, 6155–6164.

57. Wang, Y.; Jatkoe, T.; Zhang, Y.; Mutch, M.G.; Talantov, D.; et al.Gene expression profiles and molecular markers to predict recur-rence of Dukes’ B colon cancer. J. Clin. Oncol. 2004, 22, 1564–1571.

58. Ritter, C.A.; Arteaga, C.L. The epidermal growth factor receptor-tyrosine kinase: a promising therapeutic target in solid tumors.Semin. Oncol. 2003, 30 (1 Suppl. 1), 3–11.

59. Cunningham, D.; Humblet, Y.; Siena, S.; Khayat, D.; Bleiberg, H.;et al. Cetuximab monotherapy and cetuximab plus irinotecan in

irinotecan-refractory metastatic colorectal cancer. N. Engl. J. Med.2004, 351, 337–345.

60. Fruehauf, J. EGFR function and detection in cancer therapy. J.Exptl. Ther. Oncology 2006, 5, 231–246.

61. Susman, E. Rash correlates with tumour response after cetuximab.Lancet Oncol. 2004, 5, 647.

62. Gebhardt, F.; Burger, H.; Brandt, B. Modulation of EGFR genetranscription by secondary structures, a polymorphic repetitive se-quence and mutations–a link between genetics and epigenetics.Histol. Histopathol. 2000, 15, 929–936.

63. Zhang, W.; Stoehlmacher, J.; Park, D.J.; Yang, D.; Borchard, E.;et al. Gene polymorphisms of epidermal growth factor receptor andits downstream effector, interleukin-8, predict oxaliplatin efficacy inpatients with advanced colorectal cancer. Clin. Colorectal Cancer2005, 5, 124–131.

64. Watson, C.J.; Webb, N.J.; Bottomley, M.J.; Brenchley, P.E. Iden-tification of polymorphisms within the vascular endothelial growthfactor (VEGF) gene: correlation with variation in VEGF protein pro-duction. Cytokine 2000, 12, 1232–1235.

65. Yang, J.C.; Haworth, L.; Sherry, R.M.; Hwu, P.; Schwartzentruber,D.; et al. A randomized trial of bevacizumab, an anti-vascular en-dothelial growth factor antibody, for metastatic renal cancer. N.Engl. J. Med. 2003, 349, 427–434.

66. Karp, J.E.; Gojo, I.; Pili, R.; Gocke, C.D.; Greer, J.; et al. Targetingvascular endothelial growth factor for relapsed and refractory adultacute myelogenous leukemias: therapy with sequential 1-beta-d-arabinofuranosylcytosine, mitoxantrone, and bevacizumab. Clin.Cancer. Res. 2004, 10, 3577–3585.

67. Lynch, H.T.; et al. Hereditary nonpolyposis colorectal carcinoma(HNPCC) and HNPCC-like families: Problems in diagnosis, surveil-lance, and management. Cancer 2004, 100, 53–64.

68. Keller, J.J.; Giardiello, F.M. Chemoprevention strategies usingNSAIDs and COX-2 inhibitors. Cancer Biol. Ther. 2003, 2, S140–145.

69. Annie, Yu H.J.; Lin, K.M.; Ota, D.M.; Lynch, H.T. Hereditarynonpolyposis colorectal cancer: preventive management. CancerTreat Rev 2003, 29, 461–470.

70. Graaf, M.R.; Beiderbeck, A.B.; Egberts, A.C.; Richel, D.J.;Guchelaar, H.J. The risk of cancer in users of statins. J. Clin. Oncol.2004, 22, 2388–2394.

71. Poynter, J.N.; et al. Statins and the risk of colorectal cancer. N.Engl. J. Med. 2005, 352, 2184–2192.

72. Erlinger, T.P.; Platz, E.A.; Rifai, N.; Helzlsouer, K.J. C-reactive pro-tein and the risk of incident colorectal cancer. JAMA 2004, 291,585–590.

73. Lipkin, S.M.; et al. The MLH1 D132H variant is associated withsusceptibility to sporadic colorectal cancer. Nat. Genet. 2004, 36,694–699.

74. Siest, G.; et al. Pharmacogenomics and drug response in cardio-vascular disorders. Pharmacogenomics 2004, 5, 779–802.

75. Chasman, D.I.; et al. Pharmacogenetic study of statin therapy andcholesterol reduction. JAMA 2004, 291, 2821–2827.

76. de Groot, E.; et al. Effect of pravastatin on progression and re-gression of coronary atherosclerosis and vessel wall changes incarotid and femoral arteries: a report from the Regression GrowthEvaluation Statin Study. Am. J. Cardiol. 1995,76, 40C–46C.

77. Packham, C.; et al. Statin prescribing in Nottingham general prac-tices: a cross-sectional study. J. Public Health Med. 1999, 21, 60–64.

78. van Hout, B.A.; Simoons, M.L. Cost-effectiveness of HMG coen-zyme reductase inhibitors; whom to treat? Eur. Heart J. 2001, 22,751–761.

Pharmacogenomics of Colon Cancer 639