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The Pharmacogenomics Journal (2010) 10, 191–199 & 2010 Nature Publishing Group All rights reserved 1470-269X/10 www.nature.com/tpj ORIGINAL ARTICLE

A pharmacogenetic study of docetaxel and thalidomide in patients with castration-resistant using the DMET genotyping platform

JF Deeken1, T Cormier2, The anticancer agent docetaxel shows significant inter-individual variation in 3 3 its pharmacokinetic and toxicity profile. Thalidomide is an active anticancer DK Price , TM Sissung , agent and also shows wide pharmacological variation. Past pharmacogenetic 4 2 SM Steinberg , K Tran , research has not explained this variation. Patients with prostate cancer DJ Liewehr4, WL Dahut3, enrolled in a randomized phase II trial using docetaxel and thalidomide X Miao2 and WD Figg3 versus docetaxel alone were genotyped using the Affymetrix DMET 1.0 platform, which tests for 1256 genetic variations in 170 drug disposition 1Lombardi Cancer Center, Georgetown University . Genetic polymorphisms were analyzed for associations with clinical Medical Center, Washington, DC, USA; response and toxicity. In all, 10 single-nucleotide polymorphisms (SNPs) in 2 3 Affymetrix, Inc., Santa Clara, CA, USA; Medical three genes were potentially associated with response to therapy: peroxisome Oncology Branch, National Cancer Institute, NIH, Bethesda, MD, USA and 4Biostatistics and Data proliferator-activated -d (PPAR-d), sulfotransferase family, cytosolic, 1C, Management Section, National Cancer Institute, member 2 (SULT1C2) and carbohydrate (chondroitin 6) sulfotransferase 3 NIH, Bethesda, MD, USA (CHST3). In addition, 11 SNPs in eight genes were associated with toxicities to treatment: spastic paraplegia 7 (pure and complicated autosomal recessive) Correspondence: (SPG7), CHST3, , family 2, subfamily D, polypeptide 6 Dr J Deeken, Lombardi Cancer Center, Georgetown University Medical Center, 3800 (CYP2D6), N-acetyltransferase 2 (arylamine N-acetyltransferase)(NAT2), ATP- Reservoir Road, NW, Washington, DC 20007, binding cassette, sub-family C (CFTR/MRP), member 6 (ABCC6), ATPase, USA. Cu þþtransporting, alpha polypeptide (ATP7A), cytochrome P450, family 4, E-mail: [email protected] subfamily B, polypeptide 1 (CYP4B1) and 10 (sodium/bile acid family), member 2 (SLC10A2). Genotyping results between drug metabolizing and transporters (DMET) and direct sequencing showed 496% of concordance. These findings highlight the role that non- CYP450 metabolizing enzymes and transporters may have in the pharma- cology of docetaxel and thalidomide. The Pharmacogenomics Journal (2010) 10, 191–199; doi:10.1038/tpj.2009.57; published online 29 December 2009

Keywords: pharmacogenomics; docetaxel; thalidomide; prostate cancer

Introduction

Individualizing therapy for patients who are treated with pharmaceutical agents is an overarching goal of basic and clinical research in this first part of the twenty-first century. In no area of medicine is this goal more critical than in medical oncology. Genetic variation in genes encoding proteins involved in Received 24 November 2008; revised 16 October 2009; accepted 1 November 2009; the metabolism and transport of drugs may account for some of the wide published online 29 December 2009 variation observed in the response to, and toxicity from, anticancer agents. Pharmacogenetics of docetaxel and thalidomide JF Deeken et al 192

Pharmacogenetic research holds the promise of reducing the sequencing of specific fragments. This work is unpredictable inter-individual variability in the efficacy and specialized and labor intensive, which in turn limits the toxicity from using drugs. institutions and investigators who can pursue genotyping Enzymes and transporters mediate the absorption, dis- and pharmacogenetic research. One of the main obstacles tribution, metabolism and of endogenous as well facing pharmacogenetic researchers is the lack of a proven, as exogenous substrates, including drugs. However, for only scalable genotyping technology that screens for SNPs in a relatively small number of absorption, distribution, meta- multiple genes with sufficient sensitivity and high-through- bolism and excretion genes have single nucleotide polymor- put capabilities to be useful in translational and clinical phisms (SNPs) and other genetic variations been identified research. The new Affymetrix drug-metabolizing that mediate the efficacy and toxicity of chemotherapy drugs. and transporter (DMET) genotyping platform (Affymetrix, These include irinotecan and UGT1A1, 6-mercaptopurine and Inc., Santa Clara, CA, USA) screens for 1256 genetic vari- thiopurine methyltransferase, 5-fluoruracil and dihydropyrimidine ations in 170 absorption, distribution, metabolism and dehydrogenase and tamoxifen and cytochrome P450, family 2, excretion genes, including 50 CYP450 genes, 72 non-CYP subfamily D, polypeptide 6 (CYP2D6).1 genes, 39 transporters and 9 other proteins involved in drug Docetaxel (Taxotere) is a semisynthetic taxane that is disposition (Table 1).24 We tested this new genotyping currently used as first-line therapy in castration-resistant platform, termed DMET, using patient samples and clinical prostate cancer (CRPC). It is thought that the major route of data from our previously completed phase II trial in patients metabolism is through the CYP3A family of enzymes.2–4 The with CRPC to analyze the pharmacogenetics of thalidomide pharmacokinetic profile of docetaxel shows wide inter- and docetaxel. individual variability, with measures such as clearance and area under the concentration curve varying as much as sixfold between patients.5,6 Even in an ethnically homo- genous patient population, variability is as high as 3.5-fold.7 Patients and methods Research using probes of CYP3A4 activity have been unsuccessful in explaining this large variation.7–10 In fact, Patients the activity of CYP3A4/3A5 accounts for as little as two- Patients diagnosed with CRPC were enrolled in a rando- thirds of the inter-individual variable pharmacology of the mized phase II clinical trial comparing docetaxel to drug.6,11 This led researchers to look at other metabolizing docetaxel with thalidomide. The results of this trial have enzymes, including glutathione-S-transferases, as well as been previously reported.25 In brief, 74 patients with CRPC drug transporters such as ABCB1 (P-glycoprotein), to help were enrolled with a 2:1 randomization to receive either explain this large variability in drug pharmacology.11–14 docetaxel and thalidomide, or docetaxel alone. Docetaxel The pharmacogenetic factors mediating docetaxel pharma- was dosed at 30 mg m–2 weekly for 3 weeks followed by a cokinetics have not been fully characterized.14 We have 1-week rest. For patients assigned to the combination arm, previously proposed that a more comprehensive analysis they received docetaxel on the same schedule, as well as into the expression and genetic polymorphisms of multiple thalidomide at 200 mg orally each day. In all, 49 patients drug enzymes and transporters may improve our under- were in the combination arm and 25 patients were in the standing of the of docetaxel.15 control arm.25 The study was approved by the institutional Thalidomide, a potent teratogen that causes dysmelia in review board of the National Cancer Institute. , has been approved for the treatment of multiple Response to treatment was determined using the prostate- myeloma and is under active clinical analysis for its use in specific antigen Working Group consensus criteria of a drop treating other malignancies. The anticancer mechanism of in serum prostate-specific antigen of X50% with no other action of thalidomide is complex and has not yet been fully evidence of disease progression. Toxicities were graded using charecterized. In vitro data suggest that it inhibits angiogen- the Common Toxicity Criteria (version 2.0) of the Cancer esis. Non-enzymatic spontaneous hydrolysis is a major route Therapy Evaluation Program/National Cancer Institute. For of metabolism, with at least 12 different products identified this pharmacogenetic study, only adverse reactions graded by this process.16 However, enzymatic activation of these as 3 (no grade 4 adverse events were experienced on this hydrolysis products is necessary for thalidomide to exert its trial) and identified as being possibly, probably or definitely anticancer activity.17–19 Two metabolites, 5-hydroxythalido- related to study medications were included. Events of bone mide and 50-hydroxythalidomide, are formed by CYP2C19 pain, which is common in this patient population, as well and to a lesser extent CYP2B6.20 CYP1A1 may have a role in as events of hyperglycemia (which were likely caused by parent drug activation.21 Other metabolism products, the use of as a docetaxel pre-medication) including dihydroxylated and glucuronide conjugates, have were not included. Finally, events of venous been found in animal models.22 Past pharmacogenetic were not included because the trial was modified to add low- studies analyzing whether SNPs in CYP2C19 accounted for molecular-weight to patients receiving thalidomide the inter-individual variation in pharmacokinetics and after an interim analysis found a high rate of such events toxicities have been inconclusive.19,23 early in the trial. Toxicity was used as a pharmacodynamic Testing for the presence of genetic variations and SNPs is measure as well as a surrogate marker for drug pharmaco- typically performed by DNA amplification using PCR and kinetics as, at least in the case of docetaxel, clearance and

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Table 1 Genes included in the DMET 1.0 panel

P450 Enzymes Non-P450 enzymes Transporters Other

CYP1A1 CYP4F2 ADH1A CROT NQO1 ABCB1 SLC15A2 AHR CYP1A2 CYP4F3 ADH1B DYPD SULT1A1 ABCB4 SLC16A1 NR1I2 CYP1B1 CYP4F8 ADH4 EPHX1 SULT1A2 ABCB7 SLC19A1 NR3C1 CYP2A13 CYP4Z1 ADH5 FMO1 SULT1A3 ABCB11 SLC22A1 PPARD CYP2A6 CYP46A1 ADH6 FMO2 SULT1B1 ABCC1 SLC22A2 PPARG CYP2A7 CYPA1 ADH7 FMO3 SULT1C1 ABCC2 SLC22A3 RALBP1 CYP2B6 CYP51A1 ALDH1A1 FMO4 SULT1C2 ABCC3 SLC22A4 RPL13 CYP2B7P1 CYP7A1 ALDH2 FMO5 SULT1E1 ABCC4 SLC22A5 SPG7 CYP2C9 CYP7B1 ALDH3A1 FMO6 SULT2A1 ABCC5 SLC22A6 XDH CYP2C8 CYP8A1 AOX1 GSTA1 SULT2B1 ABCC6 SLC22A8 CYP2C19 CYP8B1 COA GSTA2 SULT4A1 ABCG2 SLC28A1 CYP2C18 CYP11A1 CHST1 GSTA3 TPMT ATP7A SLC28A2 CYP2D6 CYP11B1 CHST2 GSTA4 UGT1A1 ATP7B SLC28A3 CYP2E1 CYP11B2 CHST3 GSTA5 UGT1A3 SLC5A6 SLC29A1 CYP2F1 CYP17A1 CHST4 GSTM2 UGT1A4 SLC7A5 SLC29A2 CYP2J2 CYP19A1 CHST5 GSTM3 UGT1A6 SLC7A7 SLCO1A2 CYP281 CYP20A1 CHST6 GSTM4 UGT1A7 SLC10A1 SLCO1B1 CYP3A4 CYP21A2 CHST7 GSTO1 UGT1A8 SLC10A2 SLC1B3 CYP3A43 CYP24A1 CHST8 GSTP1 UGT2A1 SLC13A1 SLCO2B1 CYP3A5 CYP26A1 CHST9 HNMT UGT2B11 SLC15A1 CYP3A7 CYP26C1 CHST10 MAOB UGT2B15 CYP4A11 CYP27A1 CHST11 NAT1 UGT2B28 CYP4B1 CYP27B1 CHST13 NAT2 UGT2B4 CYP4F11 CYP39A1 COMT NNMT UGT8 CYP4F12 POR

Abbreviations: DMET, drug-metabolizing enzymes and transporters; SNP, single-nucleotide polymorphism. The DMET assay and panel genotype samples for variations in 170 drug disposition genes, including 50 CYP450 genes, 72 non-CYP genes, 39 transporters and 9 other proteins involved in drug disposition. Assay genotyping identifies homozygote wild-type, homozygote variant or heterozygote alleles in 1256 SNPs and other genetic variation sites in these genes. pharmacokinetics have been previously found to correlate Direct genotyping well with drug toxicity.26–28 Genotyping was conducted through direct sequencing for variants in eight genes that were previously thought to correlate with prostate cancer risk or the pharmacology of Patient samples docetaxel and thalidomide (Table 5). PCR was performed on For 47 of the 74 originally enrolled patients, stored buffy gene fragments for each variant site using primers and PCR coat samples were archived and available for DNA extrac- conditions as previously performed for CYP17,29 CYP2C19,30 tion. This included 33 patients on the combination arm of CYP1B1,31 CYP3A5,32 ABCB133 and ABCG2.34 A50-mlreac- the trial and 14 patients on the docetaxel-alone arm. Blood tion was prepared for PCR amplification. The reaction was collected from patients, and the buffy coat layers were consisted of 1 Â PCR buffer, 2 mmol l–1 of each of the four resuspended and frozen in Trizol (Invitrogen, Carlsbad, CA, deoxynucleotide triphosphates, 1.5 mmol l chlor- USA). Genomic DNA was extracted from serum or white ide, 20 mmol l–1 of the forward and reverse primers and 1 unit blood cell buffy coat layers of whole blood from patients of Platinum Taq DNA polymerase. Direct nucleotide sequen- using either the QiAamp Ultrasens Viral DNA kit (serum) or cing was performed using the dRhodamine Terminator Cycle the QIAamp DNA blood kit (buffy coat) as described by the Sequencing Ready Reaction kit or the Big Dye Terminator manufacturer (Qiagen, Valencia, CA, USA). Cycle Sequencing Ready Reaction kit version 1.1 on an ABI 310 genetic analyzer (Applied Biosystems, Foster City, Preparation and processing of the drug-metabolizing enzyme CA, USA). The CYP17 polymorphism was analyzed using the and transporter platform (DMET) PCR–restriction fragment length polymorphism assay. For DNA processing and genotype identification for each peroxisome proliferator-activated receptor-d (PPAR-d), geno- patient sample were performed using the Affymetrix DMET mic DNA containing the 798T4C SNP site was amplified platform as recently described.24 Genotypes were deter- using PCR. Primer sequences were as follows: F1, 50-AGCAAC mined for each SNP site, reported as homozygous wild-type, ACTCACCGCCGTGTG-30 and R1, 50-ACCTCTGACATCCCCA heterozygous, homozygous variant or ‘no call’. Four samples TCCCTT-30. After an initial denaturation at 94 1C for 5 min, were run in duplicate and the repeatability from both 40 cycles of amplification with denaturation at 94 1C for 30 s, experiments was compared. annealing at 64 1C for 30 s and extension at 72 1C for 30 s was

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Table 2 Patient characteristics Table 3 Incidence of adverse reactions

No. of patients 47 Adverse reaction No. of patients Incidence (%)

Age, yearsa 66.1 (41–79) Constitutional Fatigue 3 6 Race Myalgias/weakness 3 6 Caucasian 43 Allergic reaction 2 4 African American 3 Asian 1 Cardiovascular Arrhythmia 2 4 a Gleason at diagnosis 7.9 (6–10) Chest pain/ischemia 2 4 Baseline PSAa 252 (5–1068) Neurological b Trial arm 1 2 Docetaxel 14 4 9 Docetaxel and thalidomide 33 Hallucinations 1 2 Seizure 1 2 Response Docetaxel 7 (50%) Hematological Docetaxel and thalidomide 24 (73%) 2 4 Infection 7 15 aData are expressed as the median value and range. bThe original trial consisted of 25 patients receiving monotherapy, and 49 patients 2 4 receiving the combination. There were 9/24 (37.5%) responses for the single 1 2 agent, and 25/47 (53%) responses for the combination.25 We included the Bleeding 3 6 maximum number of patients from the original trail who had a sufficiently large DNA sample for analysis with DMET. Metabolism Characteristics of the 47 patients in this pharmacogenetic study. The parent study Electrolyte disturbances 9 19 enrolled 75 patients in a 2:1 randomization between the two arms. Response rates Hepatic dysfunction 3 6 using the prostate-specific antigen (PSA) Working Group consensus criteria were slightly higher in the subset of patients included in this study when compared with the patients in the parent study. Gastrointestinal Constipation 1 2 Diarrhea 3 6 performed, followed by a final extension step of 7 min at Ileus 1 2 72 1C. Direct nucleotide sequencing PCR was conducted using the Big Dye Terminator Cycle Sequencing Ready Pulmonary Reaction kit V1.1 on an ABI Prism 3130 Â l genetic analyzer Dyspnea 5 11 (Applied BioSystems). The primer sequences for these reac- Type and incidence rates of adverse reactions included in the toxicity analysis of 0 tions were as follows: F2 5 -TGGCTTTGCCGGTGAGGA the study using the National Cancer Institute (NCI) Common Toxicity Criteria TGC-30 and R2 50- GCAGTCAGCAAGGAGCCCAG-30. Geno- (version 2.0). Only adverse events graded as 3 (no grade 4 adverse reactions were typing results by direct sequencing were previously reported experienced on this trial), and those that were judged to be possibly, probably or and not found to correlate with clinical parameters.25 definitely related to study medication were included.

Statistical analysis (70 vs 67% in the parent study). Of the 14 patients who In an exploratory manner, the association between the SNP received docetaxel alone and were included in this pharmaco- parameters and clinical response or toxicity were tested genetic study, 50% had a clinical response (partial or complete using Mehta’s modification to Fisher’s exact test.35 Results of response), compared with a responserateof37%intheoverall potential interest were limited to those in which the P-value study. Of the patients who received thalidomide and docetax- was o0.01. P-values o0.001 would suggest even stronger el, 73% of the patients included in this study had a clinical association. Results are to be interpreted as hypothesis response compared with 53% in the parent trial. Whether a generating and in the context of a large number of sample was archived and was of sufficient volume to enable exploratory evaluations performed. DNA extraction was in no way related to the patient’s response on the trial, and the higher response rates in this subset of Results patients cannot be readily explained. Toxicities experienced during the trial of grade 3 are listed in Table 3, along with the Patients percentage of study patients experiencing each toxicity. Of the 75 patients in the original study, sufficient DNA was available from 47 individuals. Demographic and clinical data DNA extraction and processing of the drug-metabolizing enzyme on these patients are presented in Table 2. The proportion of and transporter platform (DMET) patients in each arm differed from the original 2:1 randomiza- Extracted DNA content from archived samples ranged tion, with slightly more patients on the combination arm between 3.0 and 930.9 ng. Although the recommended

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Frequency of Variant Alleles Table 4 Genes and SNPs correlating with clinical response 500 and toxicity 400 300 Gene SNP Variant Clinical Toxicity 200 frequency response (n=1256) 100 Number of SNPs 0 PPAR-d rs6922548 0.15 0.0011 NS < 10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 90-100 rs2016520 0.32 0.0056a NS Frequency (%) rs1883322 0.32 0.0061a NS a Figure 1 Frequency of variant alleles. The frequencies of the single- rs3734254 0.73 0.0089 NS a nucleotide polymorphism (SNP) variant alleles in the 170 genes are rs7769719 0.90 0.0055 NS graphed. The frequency of variant alleles was in general o10% (or CHST3 rs4148943 0.58 0.0001 NS 490% in those cases in which the reference ‘wild-type’ allele is the less rs4148947 0.43 0.0023 NS common one). For 373 of the 1256 SNPs, or 30% of all loci, variant rs12418 0.42 0.0005 NS allelic frequencies were between 10 and 90%. rs730720 0.40 0.0034 NS rs4148950 0.42 0.024b 0.006 b minimal DNA sample content for optimal DMET analysis rs1871450 0.44 0.048 0.006 b was 2000 ng, we obtained call rates of between 88 and 98% rs4148945 0.61 0.011 0.010 SULT1C2 rs1402467 0.22 0.0083 NS for all SNP loci for those samples containing at least 92 ng SPG7 rs2292954 0.73 NS 0.0004 of DNA. No-call rates in those samples with o92 ng (n ¼ 15) rs12960 0.82 NS 0.004 were lower, ranging between 21 and 91%. Four samples were CYP2D6 CYP2D6*19 0.48 NS 0.002 run in duplicate and the results were compared between (2539-2542del) the two genotyping results across the 1256 genetic var- NAT2 rs1799931 0.92 NS 0.003 iation sites. Repeatability of results ranged between 98.1 ABCC6 rs2238472 0.67 NS 0.006 and 99.9%. ATP7A rs2227291 0.78 NS 0.006 Single-nucleotide polymorphisms in the 170 DMET genes CYP4B1 rs4646487 0.08 NS 0.008 were identified. An SNP is defined as a genetic variant that SLC10A2 rs2301159 0.34 NS 0.010 occurs in at least 1% of a population. Frequency of the variant Abbreviations: NS, not significant (P40.05); SNP, single-nucleotide polymorphism. alleles was in general o10% (or 490% in those cases in which aResults are from analyses restricted to docetaxel and thalidomide trial arm. the reference ‘wild-type’ allele is the less common one). For bResult did not meet statistical significance as defined in the methods of Po0.01. 373 of the 1256 polymorphisms, or 30% of all loci, variant SNPs were tested for an association with clinical response and toxicities using Mehta’s modification to Fisher’s exact test. Reported genes and SNPs were limited allelic frequencies were between 10 and 90% (Figure 1). to those in which Po0.01. SNP identification is by reference sequence number. Frequency of the variant allele in this patient sample set is also listed. Single-nucleotide polymorphisms (SNPs) associated with clinical response pharmacology, and docetaxel toxicities (P ¼ 0.51). No other In an initial screening procedure that used the generalized variant in CYP3A5 or CYP3A4 was associated with toxicity Fisher’s exact test, 28 SNPs were identified in which the (data not shown). P-value was o0.05. Given the exploratory nature of this study, and to limit false-positive results, we further limited Genotyping with the drug-metabolizing enzyme and transporter reported results to those with a P-value of o0.01. Using this platform (DMET) versus direct sequencing genotyping criterion, 10 SNPs in three genes were found to be associated Genetic variations in eight genes were identified using direct with response to therapy: PPAR-d, sulfotransferase family, sequencing, and the results were compared with those results cytosolic, 1C, member 2 (SULT1C2) and carbohydrate (chon- using the DMET platform. Concordance rates between these droitin 6) sulfotransferase 3 (CHST3) (Table 4). results ranged between 96 and 100% (Table 5). All of the polymorphisms in Tables 4 and 5 were equally distributed Single-nucleotide polymorphisms (SNPs) and toxicity across the two trial arms (P40.05; generalized Fisher’s exact For toxicities from therapy, 11 SNPs in eight genes were test). It is to be noted that in cases in which there were found to be associated at a significance level of Po0.01 disagreements between the two result sets, and sufficient (Table 4). These included two SNPs in spastic paraplegia 7 DNA was available, patient samples were resequenced. In (pure and complicated autosomal recessive)(SPG7), three in each case tested, the initial results found by direct sequencing CHST3 and single SNPs in CYP2D6, N-acetyltransferase 2 were found to be in error, and the results from the DMET (arylamine N-acetyltransferase)(NAT2), ATP-binding cassette, platform processing were confirmed (data not shown). sub-family C (CFTR/MRP), member 6 (ABCC6), ATPase, Cu þþtransporting, alpha polypeptide (ATP7A), cytochrome Discussion P450, family 4, subfamily B, polypeptide 1 (CYP4B1) and solute carrier family 10 (sodium/bile acid cotransporter family), member We present the findings from an exploratory pharmaco- 2 (SLC10A2). There was no association between patients genomic study that related SNPs with the efficacy and with the CYP3A5*3C genotype, a poor metabolizing pheno- toxicity of docetaxel and thalidomide used in a clinical type previously found to be associated with docetaxel trial of patients with CRPC. These data revealed that

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Table 5 Concordance between DMET and direct sequencing is unclear as PPARd is a nuclear receptor that is responsible genotyping for the induction of genes involved in metabolic pathways as well. Gene SNP Concordance Carbohydrate (chondroitin 6) sulfotransferase 3 encodes a member of the carbohydrate sulfotransferase family, termed CYP17 A1A1 100 chondroitin 6-O-sulfotransferase, as it is known to transfer CYP2C19 *2 97 sulfate groups from 30-phosphoadenosine 50-phosphosulfate CYP2C19 *3 100 to the six position of N-acetylgalactosamine of chondroitin. CYP1B1 *3 96 42 CYP3A5 *3C 100 CHST3 is involved in extracellular matrix remodeling, and ABCB1 2677G4T96may thus be involved in invasion/metastasis, or in endo- 43 ABCG2 Q141K 100 thelial cell adhesion. It is also involved in drug meta- PPAR-d Rs2076167 100 bolism within the .44 Numerous SNPs that may affect function have been identified.45 An intriguing finding in Abbreviations: DMET, drug-metabolizing enzymes and transporters; SNP, single- nucleotide polymorphism. this study was that genetic variations in CHST3 were Individual genotyping results using DMET and direct sequencing were compared, associated with both efficacy and toxicity, increasing the and concordance between these two data sets was compared. A result of 100% likelihood that this finding of association may indicate a indicates that the results were in complete agreement. role in docetaxel or thalidomide pharmacokinetics and/or pharmacodynamics. Further analyses into this gene and its polymorphisms in three genes (that is, PPARd, SULT1C2 possible role in drug pharmacology are warranted. and CHST3) were associated with clinical outcome measures Sulfotransferase family, cytosolic, 1C, member 2 encodes a whereas polymorphisms in eight genes (that is, SPG7, sulfotransferase that is thought to be involved in the CHST3, CYP2D6, NAT2, ABCC6, ATP7A, CYP4B1 and metabolism of drugs and endogenous substrates, and is SLC10A2) were associated with toxicity. To our knowledge, expressed both intra- and extra-hepatically as well as in the these associations represent novel genes that may be related adult kidney.46,47 Genetic variation in this gene and other to either thalidomide- or docetaxel-based therapy, although cytosolic sulfotransferases were recently reported.48 this remains unclear given that no reports have indicated SULT1C2 seems to be downregulated during that any of the above genes are involved in the metabolism progression in which it may be involved with or disposition toward either agent. Moreover, the allelic hormone metabolism and detoxification reactions.49,50 variations in genes that are known to be involved in Sulfation reactions are not known to be involved in the metabolism, transport and activity of docetaxel and thali- metabolism of docetaxel or thalidomide. Given the strong domide were not associated with any of the study end extrahepatic expression of SULT1C2, it is equally likely that points. Thus, further studies are warranted to validate and SULT1C2 is involved in the bodily disposition toward these explore these relationships, and we can only speculate as to drugs through direct metabolism, or indirect metabolism of why such strong relationships exist. Although all of these other endogenous/exogenous agents that influence the genes may be related to directly, and thus activity of docetaxel and/or thalidomide. It is also possible could be related to pharmacokinetics, they also participate that these SNPs are merely related to disease state, and not in pathways that may affect drug action and could therefore drug activity. be involved in pharmacodynamic interactions as well. Single-nucleotide polymorphisms in eight genes were Finally, some of these alleles may be related to disease state found to be associated with docetaxel and thalidomide and not necessarily alter drug metabolism or disposition. toxicities. SPG7 encodes a mitochondrial metalloprotease Peroxisome proliferator-activated receptor-d, one of three protein known as paraplegin. Genetic variations in SPG7 members of the PPAR family, is a -activated transcrip- have been found in patients with spastic paraplegia, a tion factor that forms heterodimers with the retinoid degenerative neurological condition.51 A closely related X receptor, which then binds to peroxisome proliferator gene, SPG6, which is also associated with spastic paraplegia, response elements. The gene product has been identified was recently found to function as a transporter.52 CYP2D6 for its role in lipid and cholesterol metabolism,36 whereas and CYP4B1 are members of the P450 family of phase I the polymorphisms in the gene have been related to metabolic enzymes. CYP2D6 is known to be involved in the inter-individual variation in cholesterol metabolism,37 metabolism of a wide range of drugs.1 Inhibition of this insulin sensitivity,38 psychological disorders39 and reduced enzyme was not found to affect the formation of hydroxyl- height.40 Although no study has yet evaluated PPARd SNPs docetaxel, which is thought to be mediated by CYP3A4.53 in relation to antiangiogenesis therapy, PPARd gene expres- Whether this enzyme is involved in the formation of other sion is a major hub node in the angiogesis network,41 thus docetaxel metabolites is unknown. microsome opposing thalidomide action. Given that we observed studies did not find a significant role for CYP2D6 and other strong relationships with several SNPs in PPARd in only CYP enzymes in thalidomide metabolism.54 It is also those patients who received both docetaxel and thalido- possible that certain CYP2D6 alleles may confer a multi- mide, but not docetaxel alone, it seems that allelic variation ple-chemical sensitivity phenotype that increases the in PPARd may influence therapeutic efficacy of the anti- likelihood of development of toxicities after drug treatment agent, thalidomide. However, this relationship in general.55

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N-acetyltransferase 2 (arylamine N-acetyltransferase) encodes with pharmacokinetics and/or pharmacodynamics. These a polymorphic drug and xenobiotic-metabolizing enzyme exploratory studies can then be followed by high-through- that acetylates substrates. Genetic variants in NAT2 can put analyses using larger clinical sample sets.11,61 New tools, cause a ‘slow’, ‘intermediate’ or ‘rapid’ acetylator pheno- such as the DMET platform, will make pharmacogenetic type. These variants can predict efficacy and toxicity of a research available to more researchers, which may lead to wide range of drugs, including sulfasalazine56 and isonia- answering clinical questions in medical oncology and other zid.57 ABCC6 encodes a xenobiotic transporter from the C fields. This, in turn, may hasten the day when the promise subfamily of the ATP-binding cassette (ABC) family of of individualized pharmacogenetic therapy becomes a transporters. ABCC6, also termed MRP6, is known to be reality for more of our cancer patients. involved in the transport of glutathione conjugates and may mediate drug resistance to agents, such as etoposide, Conflict of interest teniposide, and daunorubicin.58 ATP7A en- codes a transporter that is characterized best for its role in TC, KT and XM are employees of Affymetrix, the manu- copper disposition, and is also thought to mediate cisplatin facturer of the DMET platform. JFD serves as a consultant to 59 and carboplatin transport and resistance. Finally, SLC10A2 -Aventis, the manufacturer of docetaxel. encodes a sodium-dependent transporter highly expressed in the ileum and liver involved in the enterohepatic circulation of bile acids.60 Polymorphisms in these enzymes Abbreviations and transporters might alter the disposition toward xeno- biotics in general, and could potentially be involved in CRPC castrate-resistant prostate cancer DMET drug-metabolizing enzymes and transporters pharmacokinetic and pharmacodynamic relationships with SNP single-nucleotide polymorphism docetaxel and thalidomide. However, as the mechanism behind these associations is unclear, it is difficult to ascertain the importance of these findings. This is the first study to report that these enzymes and Acknowledgments transporters may be involved in the disposition of docetaxel and thalidomide, and/or their metabolites. This raises the This work was supported, in part, by the Intramural Research possibility that these results are false positives and reflect Program of the NIH, National Cancer Institute, Center for Cancer only random associations that are found to be statistically Research, Bethesda, MD, USA. Partial findings contained in this significant. The risk of false-positive findings can be article were initially presented at the 2007 Annual Convention of the American Society of Clinical Oncology. common in pharmacogenetic research as more polymor- phisms, combinations of polymorphisms or phenotypes are included in studies with limited number of study subjects.61 Disclaimer Given the breath of the DMET platform, with 170 genes and 1256 polymorphisms, this was a potential shortcoming in The content of this paper does not necessarily reflect the views our study. To reduce the risk of false-positive results, we or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or restricted reported results to those in which the P-value was organizations imply endorsement by the US Government. o0.01, using Mehta’s modification to Fisher’s exact test. As an exploratory study analyzing associations between SNPs References and clinical parameters, this was meant to be hypothesis generating and provide targets for future—and larger— 1 Deeken J, Figg W, Bates SE, Sparreboom A. Toward individualized pharmacogenetic clinical studies.61 Research to confirm treatment: prediction of anticancer drug disposition and toxicity with these exploratory findings are ongoing. pharmacogenetics. Anticancer Drugs 2007; 18: 111–126. The promise of the emerging field of pharmacogenetics is 2 Sparreboom A, Van Tellingen O, Scherrenburg EJ, Boesen JJ, Huizing MT, Nooijen WJ et al. 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