Pharmacogenomics.pdf

Pharmacotherapy Self-Assessment Program, 6th Edition 39 Pharmacogenomics

Learning Objectives 1. Distinguish between the different types of mutations

(e.g., single nucleotide polymorphisms, repeat polymorphisms) associated with variations in therapeutic response and toxicity in patients who receive anti-cancer drugs.

2. Predict response and tolerability to anti-cancer drugs based on genetic and nongenetic factors.

3. Interpret pharmacogenetic data with respect to selecting appropriate anti-cancer drug therapy.

4. Modify the treatment regimen for a patient with cancer using pharmacogenetic information.

5. Assess how the use of pharmacogenetic data can improve therapeutic outcomes associated with anti-cancer drugs.

Introduction Our current approach to drug therapy is largely empiric and based on clinical studies that define the maximally tolerated dose and reasonable toxicity in a narrowly defined population. This approach typically leads to the safe and effective administration of drugs to most individuals. However, empiric therapy is more likely to lead to poor responses or exaggerated toxicities in patients receiving drugs with a small therapeutic window, such as anti-cancer drugs. Tailoring therapy using genetic information is one means to improve response and tolerability to anti-cancer drugs. Clinical pharmacogenomics is the study of the human genome for understanding the variability in response to drug therapy. Genetic variations may explain in part some of the well-documented variability of response to anti-cancer drugs. Polymorphisms are genetic variations that occur in at least 1% of the population. The most common polymorphisms are single nucleotide polymorphisms, which occur when one nucleotide base replaces another nucleotide base within the DNA sequence. These polymorphisms may be synonymous or nonsynonymous; a nonsynonymous

single nucleotide polymorphism causes a change in the amino acid and, ultimately, the protein. Other examples of polymorphisms include copy number polymorphisms, in which multiple copies of an entire gene exist; and insertions or deletions, in which DNA bases are added or removed compared with the wild-type DNA sequence. Many studies now indicate that genetic information may be predictive of response or tolerability to a specific drug or provide prognostic information regarding the disease. In the past few years, substantial progress has been made in incorporating pharmacogenetics into clinical practice. The inclusion of pharmacogenetic information in package inserts, as well as the availability of U.S. Food and Drug Administration (FDA)-approved genomic tests, demonstrates the importance of this information. These new developments have made genomic testing feasible within the clinical arena, and some insurance companies now reimburse the costs of many of these tests. However, these tests are not being used widely. One limitation to performing genetic tests is the ability to interpret and make recommendations for therapy based on the results; another is that most pharmacogenetic data are from whites; thus, less is known about other ethnicities. Still other limitations include variability between the sexes and the lack of standardization of methods for identifying specific genetic changes. For pharmacists involved in the selection and recommendation of appropriate drug therapy, a thorough understanding of pharmacogenomics and its application to drug therapy is necessary. This chapter focuses on areas of cancer therapeutics in which pharmacogenetic information has been evaluated and has proved effective in predicting clinical response or tolerability.

Tamoxifen Tamoxifen is a selective estrogen receptor modulator commonly administered to women with hormone receptorpositive breast cancer after surgery with or without adjuvant chemotherapy. The optimal duration of therapy remains poorly defined, but 5 years of therapy can reduce the relative risk of recurrence by almost 50%, and the benefits last for

Pharmacogenomics

Stacy S. Shord, Pharm.D., BCOPReviewed by Christine M. Walko, Pharm.D., BCOP; Katherine H. Chessman, Pharm.D., FCCP, BCPS, BCNSP; and Rodney Gedey, Pharm.D., BCPS

Pharmacotherapy Self-Assessment Program, 6th Edition40Pharmacogenomics

Abbreviations in This ChapterALL Acute lymphoblastic leukemiaAML Acute myeloid leukemiaATP Adenosine triphosphateCML Chronic myeloid leukemiaCYP Cytochrome P450DPD Dihydropyrimidine dehydrogenaseEGFR Epidermal growth factor receptorFISH Fluorescence in situ hybridizationIHC ImmunohistochemistryKRAS Kirsten retrovirusassociated

DNA sequencesSSRI Selective serotonin reuptake inhibitorSULT SulfotransferaseTPMT Thiopurine methyltransferaseUGT Uridine diphosphate-

glucuronosyltransferase

subvariants, have been identified to date. Phenotype prediction based on drug concentrations or genotype is common, and more than one system is described. The phenotype can be characterized as ultraextensive metabolizer, extensive metabolizer, intermediate metabolizer, or poor metabolizer. The ultraextensive metabolizers possess multiple copies of functional alleles (e.g., *1, *2) and metabolize drugs faster compared with extensive metabolizers. Intermediate metabolizers typically possess one reduced function (e.g., *3, *4, *17, *29, *41) or null allele (e.g., *5), and poor metabolizers typically possess two reduced function or null alleles. These metabolizers break down drugs slower when compared with extensive metabolizers. About 7% to 10% of whites and 2% to 5% of African Americans poorly metabolize CYP2D6 substrates. The most common alleles associated with poor metabolic status in whites are CYP2D6*3, *4, and *5, and in African Americans, they are CYP2D6*17 and *29. Two studies strongly indicate that variability in CYP2D6 activity or expression may affect the metabolism of tamoxifen. The concentrations of tamoxifen and its metabolites were measured in 12 women receiving tamoxifen therapy before and after 4 weeks of paroxetine therapy. Paroxetine is a selective serotonin reuptake inhibitor (SSRI) and a known inhibitor of CYP2D6 that is commonly prescribed to minimize hot flashes associated with tamoxifen. Endoxifen concentrations decreased significantly (from 12.4 ng/mL to 5.5 ng/mL) after paroxetine treatment. Furthermore, endoxifen concentrations decreased less in women with a variant CYP2D6 genotype (24%) compared with women with a wild-type CYP2D6 genotype (64%). In a follow-up study to validate these initial findings, plasma concentrations of tamoxifen and its metabolites were measured in 80 women 1 and 4 months after beginning adjuvant tamoxifen therapy; the concentrations of the metabolites were at steady state 4 months after starting tamoxifen. The SSRIs included paroxetine, fluoxetine, setraline, citalopram, and venlafaxine (in order of most-potent to least-potent inhibitor of CYP2D6). Genotypic analysis was performed for CYP2D6, CYP2C9, CYP3A5, and SULT1A1. Mean endoxifen concentrations were 55% lower in heterozygotes compared with homozygotes for wild-type CYP2D6; the variant alleles examined included *1 (wild-type) and *3 to *6. Furthermore, among patients homozygous for wild-type genotype, mean plasma endoxifen concentrations were 58% lower in patients taking SSRIs compared with patients not taking SSRIs. Endoxifen concentrations also were lower in women taking paroxetine compared with those taking venlafaxine, consistent with paroxetine being the most potent inhibitor and venlaxafine being the least potent inhibitor of CYP2D6 when SSRIs are compared. The mean plasma concentrations for endoxifen, 4-hydroxytamoxifen, and N-desmethyl-tamoxifen were not associated with genotypes for CYP2C9, CYP3A5, or SULT1A1. These studies demonstrate that the CYP2D6 phenotype and genotype are strongly associated with endoxifen concentrations. However, it was not clear from these studies whether these pharmacokinetic changes influence clinical outcomes because response and tolerability were not assessed. Later studies, as discussed in the following section, suggest a possible relationship

up to 15 years after discontinuing therapy. The aromatase inhibitors (e.g., anastrozole, letrozole) are reasonable alternatives to tamoxifen in postmenopausal women with hormone receptorpositive breast cancer. Many studies indicate that the aromatase inhibitors further decrease recurrence, cause fewer adverse events than tamoxifen, and may improve survival when administered sequentially with tamoxifen. However, recent data supporting an association between tamoxifen and metabolic status raise the question whether the outcomes with tamoxifen may rival the outcomes of aromatase inhibitors if the studies comparing these drugs include only extensive metabolizers of tamoxifen. Tamoxifen is a prodrug with a complex metabolic profile; it undergoes extensive phase 1 metabolism by several cytochrome P450 (CYP) enzymes, including CYP3A4 and CYP2D6. Cytochrome P450 enzymes, including CYP3A4, catalyze the metabolism of tamoxifen to N-desmethyl-tamoxifen, which is subsequently metabolized by CYP2D6 to endoxifen. The metabolism of tamoxifen to 4-hydroxytamoxifen is also catalyzed by CYP2D6. These metabolites can undergo additional metabolism by sulfotransferase-1A1 (SULT1A1) and by uridine diphosphate-glucuronosyltransferase (UGT), specifically UGT2B7 or UGT2B15. Tamoxifen and its metabolites, 4-hydroxytamoxifen and endoxifen, can bind to estrogen receptors and suppress estrogen-dependent cell proliferation. Both metabolites demonstrate greater affinity for the estrogen receptor compared with tamoxifen and greater potency in inhibiting estrogen-dependent cell proliferation. Endoxifen concentrations exceed those of 4-hydroxytamoxifen by 10-fold, suggesting that endoxifen is the metabolite responsible for the beneficial clinical outcomes associated with tamoxifen.

CYP2D6 Polymorphisms The CYP2D6 enzyme accounts for about 5% of total hepatic CYP protein and metabolizes about 30% of drugs. About 70 CYP2D6 variant alleles, together with additional


between CYP2D6 and clinical outcomes in women receiving adjuvant tamoxifen.

Genotype and Clinical Outcomes Using paraffin-embedded tissue blocks from 223 women with hormone receptorpositive breast cancer, the association between the CYP2D6 genotype and tamoxifen efficacy was evaluated. The DNA for CYP2D6*4, CYP2D6*6, and CYP3A5*3 was amplified in at least 190 tissue blocks. Women with the CYP2D6*4/*4 genotype had shorter relapse-free times and disease-free survival rates, but they did not have significantly different overall survival rates. However, more women with this genotype had lymph nodepositive disease, which is a negative prognostic factor for women with early-stage breast cancer. These women also experienced less-severe hot flashes, an effect associated with tamoxifen. No CYP2D6*6 alleles were detected, and CYP3A5*3 alleles were not associated with tamoxifen efficacy. In an updated analysis, the investigators examined the medical records to determine whether CYP2D6 inhibitors were prescribed with tamoxifen. The women were characterized as extensive metabolizers, intermediate metabolizers, or poor metabolizers. Extensive metabolizers were women without the CYP2D6*4 allele or coadministered a CYP2D6 inhibitor. Women with decreased metabolism, including intermediate and poor metabolizers, experienced a shorter time to recurrence and poorer relapse-free survival; poor metabolizers demonstrated worse outcomes compared with intermediate metabolizers. These initial findings suggest that the changes observed in mean endoxifen concentrations in association with the CYP2D6 genotype influence both response and tolerability. Four additional studies were published that examined the CYP2D6 genotype as a marker of response to tamoxifen. The first study enrolled 206 women receiving tamoxifen and 280 women not receiving therapy. Women heterozygous for the CYP2D6*4, *5, *10, and *41 alleles and taking tamoxifen had increased disease recurrence, shorter relapse-free periods, and poorer event-free survival rates than women not receiving therapy; these results support the data from the first study described. Furthermore, a positive association between the CYP2C19 genotype and tamoxifen outcomes was found. Women heterozygous for CYP2C19*17 had a more favorable outcome compared with women heterozygous for *1, *2, and *3 alleles; of note, the *17 allele is associated with increased catalytic activity. One retrospective analysis supported an association between the CYP2D6 genotype and higher disease-free survival rates, whereas two retrospective studies did not find an association between the CYP2D6 genotype and disease recurrence or survival in women taking tamoxifen. Conflicting data are likely caused by differences in study design and patient population and should be confirmed prospectively. Additional limitations to these studies are the small number of alleles evaluated and the lack of consistent reporting of concomitant drugs that may also affect endoxifen levels. Associations between survival and genotype of other phase 1 and two phase 2 metabolic enzymes in women taking tamoxifen were also examined in some of these latter studies. The enzymes UGT2B15 and SULT1A catalyze the glucuronidation and sulfation of 4-hydroxytamoxifen, respectively. The most common variant allele, UGT2B15*2,

is associated with increased enzyme activity, whereas the most common variant allele, SULT1A*2, is associated with a 2-fold reduced metabolic capacity compared with the wild-type allele. It was anticipated that these polymorphisms would affect the therapeutic response associated with tamoxifen because further metabolism of 4-hydroxytamoxifen would terminate its effects on estrogen receptors. Women taking tamoxifen and identified as homozygous for SULT1A1*2 had a greater risk of death than women identified as heterozygous or with homozygous wild-type genotype. Similarly, women identified as carriers of UGT2B15 high-activity genotypes experienced more disease recurrence and shorter survival times than those without the genotype. Women homozygous for SULT1A1 trended toward improved disease-free recurrence compared with women homozygous for the wild type, confirming earlier findings. Furthermore, women homozygous for the CYP3A5*3 allele who received tamoxifen for 5 years had longer recurrence-free survival than those treated for a shorter duration; however, this improvement was not significant for women receiving tamoxifen for 2 years. These retrospective analyses support an association between phase 2 metabolic status and outcomes of tamoxifen therapy, but additional data are warranted to confirm these preliminary findings, especially in light of recent data indicating that endoxifen is a more potent active metabolite compared with 4-hydroxytamoxifen. Prospective studies in which patients are treated based on genotype are warranted to confirm these data before metabolic status for phase 2 enzymes can be considered in treatment decisions.

Clinical Testing In 2006, the FDA supported the inclusion of data regarding an association between the CYP2D6 genotype and an increased risk of disease recurrence in the labeling of tamoxifen; however, no consensus was reached regarding a recommendation for genetic testing. A DNA microarray currently available from Roche is the first FDA-cleared in vitro diagnostic test for CYP2C19 and CYP2D6. Genotype information for 29 alleles is compiled to predict an individuals metabolic capacity based on published reports. The specificity of the test is 100% for the wild-type alleles, and the sensitivity is 99% for CYP2D6 and CYP2C19 alleles. The genotype is reported as an extensive, intermediate, or poor metabolizer. This microarray could be used to determine CYP2D6 metabolic capacity before administering tamoxifen, if prospective studies ultimately indicate that determining a womans genotype before receiving tamoxifen improves long-term outcomes. Although data support a relationship between genotype and recurrence, no prospective study provides evidence that genotyping before initiating tamoxifen therapy improves outcomes compared with no genotyping. Furthermore, it is not known whether genetic testing will improve outcomes in postmenopausal women receiving tamoxifen compared with women receiving aromatase inhibitors. Additional concerns stem from the limited genotypes examined retrospectively; more than 70 variant CYP2D6 alleles have been identified to date, and although CYP2D6 substrate specificity is dependent on race, the inclusion of different ethnicities in these analyses has been limited.


Irinotecan Irinotecan is a camptothecin analog that inhibits topoisomerase-1. This anti-cancer drug, which is commonly used to treat colon cancer in combination with fluorouracil or cetuximab, may also be considered for other solid tumors, including nonsmall cell lung cancer. The dose-limiting adverse effects of irinotecan include diarrhea and neutropenia. Irinotecan undergoes metabolism by carboxylesterases to its active metabolite SN-38. This metabolite undergoes further metabolism by UGT to a more soluble and polar metabolite SN-38G that facilitates its elimination in the bile and urine. The main glucuronosyltransferase responsible for the glucuronidation of SN-38 is UGT1A1. Several polymorphisms are described for this enzyme, but the most common polymorphism is UGT1A1*28. This variant allele contains seven tandem repeats in the promoter region of the gene compared with six tandem repeats in the wild-type allele UGT1A1*1. The promoter region of the gene is the regulator that facilitates transcription. The UGT1A1*28 allele is associated with reduced gene expression and catalytic activity in human liver tissue.

UGT Polymorphisms and Toxicity Many studies indicate that the incidence of grade 3 or 4 toxicity with irinotecan treatment is higher in carriers of the UGT1A1*28 allele. In one study, 20 patients received irinotecan 300 mg/m2 over 90 minutes every 3 weeks. The allele frequencies were 0.375 and 0.625 for UGT1A1*28 and UGT1A1*1, respectively. Three patients identified as homozygous or heterozygous for the variant allele developed grade 3 or 4 neutropenia or diarrhea compared with no patient identified as homozygous wild-type genotype. The more severe toxicity was attributed to decreased glucuronidation in the patients with the UGT1A1*28 allele. In a larger study, the prevalence of grade 4 neutropenia was 50% for patients homozygous for the variant allele, 12.5% for patients heterozygous for the variant allele, and 0% for patients homozygous for the wild-type allele. Furthermore, pretreatment bilirubin concentrations correlated with the UGT1A1 genotype; because this enzyme is responsible for the conjugation of bilirubin, these data suggest that bilirubin may be used as a phenotypic marker for genotype. Five additional studies confirm these data by indicating that the variant allele is associated with greater toxicityeither neutropenia or diarrhea. In contrast, two studies indicate that the UGT1A1 genotype does not predict toxicity. Different patient populations and chemotherapy regimens may be responsible for the apparent differences in outcomes, but the consensus appears to be that the UGT1A1 genotype is associated with toxicity. Only one study to date has evaluated the association between UGT1A1*28 and toxicity, response rate, and overall survival. The incidence of neutropenia was 4-fold higher in heterozygotes of UGT1A1*28, and the incidence of neutropenia and diarrhea was 8.6-fold and 4.1-fold higher in homozygotes of UGT1A1*28. A univariate analysis indicated that carriers of this allele showed a trend toward poorer overall survival; however, this association was not confirmed as part of the multivariate analysis. No relationship between

genotype and response rate was found. These studies together support an association between UGT1A1*28 and grade 3 or 4 toxicities; however, a relationship between efficacy and genotype has not been clearly identified. A recent meta-analysis suggested that the association between the UGT1A1 genotype and grade 3 or 4 toxicity may only be evident when a patient receives doses of irinotecan greater than 150 mg/m2. This meta-analysis included all of the individual studies described previously. The analysis indicated that patients who were homozygous for the UGT1A1*28 allele and who received doses greater than 250 mg/m2 were more likely to develop hematologic toxicity than patients receiving lower doses of irinotecan. In contrast, patients homozygous for this variant allele who received doses less than 150 mg/m2 were not more likely to develop hematologic toxicity. No association between UGT1A1 genotype, dose, and diarrhea was observed. These data suggest that the UGT1A1*28 genotype may only be associated with hematologic toxicity at higher doses. Therefore, genotyping may not be beneficial in predicting the development of hematologic toxicity in patients receiving lower weekly doses.

Clinical Testing In 2005, irinotecan product information was revised to include a description of the prevalence of UGT1A1 polymorphisms, the increased risk of neutropenia with these polymorphisms, and a recommendation to start treatment with a lower dose in these patients. A one-level dose reduction (about a 20% dose reduction) is recommended for patients homozygous for the UGT1A1*28 allele. In August 2005, the FDA approved a pharmacogenetic test for UGT1A1. This test detects and identifies mutations in the UGT1A1 gene. The test can be used as an aid to individualize the dose of irinotecan for a specific patient; however, it is not clear how this information should be used. Considerations regarding therapy should include treatment intent (curative vs. palliative), alternative therapies, and clinical evidence regarding the outcomes associated with lower doses. Furthermore, the racial distribution in studies is not always provided within the publications; thus, it is not known if this association is applicable to all races.

Fluorouracil Fluorouracil is an integral part of chemotherapy regimens for gastrointestinal, breast, cervical, and head and neck cancers. The most common grade 3 or 4 toxicities include hematologic, dermatologic, and gastrointestinal toxicities. Fluorouracil is a pyrimidine analog that is inactivated by the polymorphic enzyme dihydropyrimidine dehydrogenase (DPD); this catabolism is the initial and rate-limiting step in the inactivation of fluorouracil and metabolizes more than 80% of fluorouracil. Its oral analog, capecitabine, also is inactivated by DPD. The remaining portion of the drug undergoes metabolism to form a monophosphate, which forms a complex that impairs DNA synthesis and is responsible for the antiapoptotic and cytotoxic effects associated with fluorouracil.


DPD Polymorphisms The catalytic activity of DPD varies by at least 20-fold, and low activity substantially limits the inactivation of fluorouracil. More than 55 genetic variations, including synonymous and nonsynonymous single nucleotide polymorphisms and gene deletions, have been identified; however, most variations do not have functional consequences. The most common polymorphism, DYPD*2A, accounts for 40% to 50% of patients with reduced or no DPD activity. The polymorphism leads to the transcription of a nonfunctional protein. Subsequently, the elimination half-life of fluorouracil is increased in patients heterozygous for the DYPD*2A allele. Initial data suggest that DPD deficiency may be associated with improved response or decreased tolerability; however, a molecular basis for reduced activity is only defined in about 57% of patients. Sex and racial differences in DPD activity have been reported. Women experience greater toxicities compared with men after receiving fluorouracil; it appears that DPD activity in the tumor tissue is lower in women compared with men. Reduced DPD activity is reported in 3% to 5% of whites and in 8% of African Americans. Furthermore, mean DPD activity is lower in African Americans and higher in Asians compared with whites. Evaluation of the literature also indicates that single nucleotide polymorphisms and haplotype distributions are dependent on race and ethnicity. However, these comparisons should be interpreted with caution because no consensus exists regarding the definition of deficiency.

DPD Deficiency and Toxicity Because more than 30% of patients receiving fluorouracil describe severe toxicity, a causal relationship between DPD deficiency and toxicity after fluorouracil has been examined for about 2 decades. Reduced DPD activity is associated with increased toxicity with fluorouracil and capecitabine because of increased accumulation of the pyrimidine analogs. Three cohort studies and five case reports demonstrated a correlation between specific DYPD mutations and grade 3 or 4 toxicities after fluorouracil. The most common variant, DYPD*2A, should not be singled out; one case series indicated normal DPD activity was associated with lethal toxicity. The DYPD*2A was found in only 2 of 93 patients and was not associated with toxicity. Four additional studies found that DPD deficiency was associated with increased toxicity with fluorouracil or capecitabine. In these studies, DPD deficiency was defined using plasma dihydrouracil-to-uracil ratio alone or in combination with genotype. Of interest, some patients included in these studies who experienced severe toxicity were not carriers of the DYPD*2A allele. Overall, ample evidence suggests a strong relationship between fluorouracil exposure, DPD activity, and severe hematologic and gastrointestinal toxicities. Mild toxicities have been associated with high and low DPD activity. Grade 1 or 2 neutropenia was associated with elevated DPD activity in peripheral blood mononuclear cells, whereas grade 1 or 2 dermatologic toxicity was associated with reduced DPD activity. These data suggest that tailoring fluorouracil therapy may be difficult because different toxicities are associated with either high or low DPD activity.

One prospective study supports tailoring fluorouracil therapy based on genetic factors. The study was conducted to determine whether genetic factors would be helpful in individualizing fluorouracil therapy. Toxicity was linked to a poor performance status of the patient, a low dihydrouracil-to-uracil ratio, and DYPD*2A in patients receiving fluorouracil as first-line therapy for advanced colorectal cancer. Prospective studies designed to tailor therapy using these factors are needed before screening for DPD deficiency is widely used in the clinical setting.

DPD Expression and Efficacy Dihydropyrimidine dehydrogenase expression may be associated with the clinical response of solid tumors to fluorouracil. Gene expression, protein expression, and activity levels of DPD alone and in combination with various enzymes involved in the metabolism and response to fluorouracil have been examined in tissue blocks. Lower intratumoral DPD expression or activity was associated with improved response to fluorouracil in patients with head and neck, breast, gastric, and colon cancers; however, the findings vary from study to study and should be interpreted with caution. Furthermore, relative gene or protein expression does not appear to correspond with DPD activity, further limiting the interpretation of these studies.

Clinical Testing Screening for DPD deficiency includes both phenotypic and genotypic methods. The most promising assay for clinical use appears to be the dihydrouracil-to-uracil ratio because this ratio corresponds to fluorouracil clearance, and the assay has been streamlined to improve clinical usefulness. This ratio also corresponds with fluorouracil plasma concentrations and toxicity in patients receiving adjuvant therapy for colorectal cancer. Genotypic assays include measurement of DYPD messenger RNA relative expression or copy number, but it is not clear whether relative gene expression corresponds with DPD enzymatic activity. These methods focus on identifying the most common polymorphisms, but they require improvement before clinical application can be considered. It is not yet possible to screen patients for DPD deficiency. Testing methods will need to be validated in prospective studies and demonstrate a positive impact on clinical outcomes without a negative economic impact. Empiric dosing, followed by dose modifications for myelosuppression or hepatic impairment, remains the standard of care.

Mercaptopurine Mercaptopurine is an oral purine analog commonly prescribed to patients with acute lymphoblastic leukemia (ALL) in combination with methotrexate as part of maintenance therapy; its dose-limiting toxicities are neutropenia and anemia. Mercaptopurine undergoes metabolism by a series of nucleotide enzymes to several phosphorylated metabolites that are eventually incorporated into RNA and DNA. The cytosolic enzyme thiopurine methyltransferase (TPMT) catalyzes the inactivation of mercaptopurine and


produces an inactive metabolite, 6-methylmercaptopurine. S-adenosylmethionine serves as the methyl donor of the reaction. This enzyme also catabolizes mercaptopurine nucleotides to inactive methylated nucleotides.

TMPT Polymorphisms Thiopurine methyltransferase activity has large variability in whites, with a trimodal distribution observed in most individuals possessing high activity. About 10% of individuals possess intermediate activity, and 1% have low activity. These individuals may be characterized as extensive, intermediate, or poor metabolizers. Extensive metabolizers possess two wild-type alleles, intermediate metabolizers possess one variant allele associated with reduced metabolic capacity, and poor metabolizers possess two variant alleles associated with reduced metabolic capacity. The gene is inherited as an autosomal codominant trait, and at least 19 variants have been identified to date. The most common polymorphisms in whites are TPMT*2 and TMPT*3A alleles, which account for up to 95% of reduced enzymatic activity. The frequencies of mutations and the most common variant alleles differ by ethnic population. For example, Indian Asians have a lower frequency of variant TPMT alleles compared with whites, and all variant alleles identified to date are TPMT*3A. In comparison, African Americans have a frequency of variant alleles similar to whites, but all variant alleles in identified to date are TPMT*3C. The contribution of the remaining variant alleles to reduced activity is not defined in whites or other populations. Other variants identified include variable numbers of tandem repeats in the 5-flanking promoter region of the gene. The impact of these tandem repeats on enzymatic activity is not clear; one study demonstrates minimal declines in activity, whereas other studies demonstrate no change in activity.

TMPT Deficiency and Clinical Outcomes It is documented that reduced TPMT activity is associated with an exaggerated or poor response dependent on genotype response or severe hematologic toxicity. Individuals heterozygous for one variant allele tend to have improved response rates compared with individuals homozygous for the wild type. Studies indicate that individuals labeled as extensive metabolizers of standard doses of mercaptopurine are at about a 3-fold greater risk of relapse compared with intermediate metabolizers (heterozygotes) with standard doses. It appears individuals identified as heterozygotes for a variant TPMT allele may demonstrate improved outcomes compared with homozygotes for the wild-type allele using standard doses based on one study. Individuals homozygous for a variant allele tend to have a greater risk of severe myelosuppression when receiving standard doses. The total population in one study received 89% of the planned maintenance dose. The planned maintenance dose did not differ between patients identified as homozygous wild type or heterozygous for a variant allele associated with reduced TPMT activity, but the planned maintenance dose was reduced to 53% secondary to myelosuppression in patients identified as homozygous variant allele. About 12% of patients were identified as carriers of a variant allele. Because dose intensity is strongly associated with outcomes, it is possible

that patients homozygous for a variant allele are more likely to have poorer outcomes. Although response rates were not examined in this study, the known relationship between dose intensity and outcomes leads to the conclusion that multiple dose interruptions that occur in patients with TPMT deficiency receiving standard doses lead to poorer outcomes. Therefore, these patients should receive lower doses to permit the maintenance of dose intensity and increase the likelihood of improved outcomes. The lower dose also permits patients to receive full doses of other myelosuppressive drugs. Dosing adjustments before beginning therapy are recommended for individuals with TPMT deficiency. For intermediate metabolizers, the dose should be reduced to 65% of the standard dose; for poor metabolizers, the dose should be reduced to 6% to 10% of the standard dose. These adjustments are based on the reported percentage of weeks that patients heterozygous or homozygous for a variant allele were able to tolerate standard therapy with mercaptopurine as part of maintenance therapy for ALL. If an individual patient tolerates the dose, the dose can be gradually increased to maximize response without causing severe neutropenia. Reports from small numbers of patients suggest a relationship between TPMT activity and long-term adverse effects. Thiopurine methyltransferase deficiency is associated with an increased incidence of brain tumors and secondary leukemias. In a study conducted at St. Jude Childrens Research Hospital, the 8-year cumulative incidence of brain tumors was 43% among children with TPMT deficiency compared with 8.3% among children without TPMT deficiency. Two studies indicate that TPMT activity might be lower in patients who develop secondary acute myeloid leukemia (AML) after receiving mercaptopurine for ALL. In one study, 55 patients with lower TPMT activity (defined as less than 14 units/mL of red blood cells) had a 5-year risk of AML of 9% compared with 1% for the patients with higher activity. The leukemogenic effect may also be increased in individuals with low TPMT activity when mercaptopurine is administered with other cytotoxic anti-cancer drugs.

TMPT Testing Both phenotyping and genotyping methods may be used to identify TPMT deficiency. Phenotyping assays include ex vivo measurement of thioguanine nucleotides or TPMT activity in erythrocytes. Thiopurine methyltransferase activity may be measured using radiolabeled substrates, high-performance liquid chromatography, and radioimmunoassays. Limitations include poor concordance among the different methods for measuring thioguanine nucleotides or TPMT activity in erythrocytes; one study suggested that up to a 2.6-fold difference in TPMT activity measurement is caused by the methodology. Red blood cell transfusions and concurrent drug therapy with sulfasalazine and other aminosalicylate drugs may also affect these methods. Moreover, the underlying disease may influence enzymatic activity; one study indicated that TPMT activity was higher in blasts from patients with AML compared with blasts from patients with ALL, making the definition of TPMT deficiency dependent on the disease state. However, TPMT activity still corresponded with genotype in these populations.


Transactivating mutations show a high concordance with activity assays in patients with cancer and healthy volunteers, suggesting genotype can be used in place of phenotype for detecting TPMT deficiency. Genotype methods used to detect the mutations include polymerase chain reaction, followed by restriction fragment length polymorphism or, more recently, pyrosequencing for the most common mutations. The product information for mercaptopurine summarizes the data regarding the relationship between TPMT and myelosuppression as described above. A statement included in the product information supports TPMT testing when patients experience severe myelosuppression after induction chemotherapy. Furthermore, the product information contains a statement that a patient with little or no TPMT activity is at an increased risk of severe myelosuppression and typically requires substantial dose reductions. No specific dose recommendations are provided. Overall, a strong association between low TPMT activity and myelosuppression after mercaptopurine is well documented. Limitations in TPMT testing include lack of concordance between the various methods and lack of validation in different ethnic populations. Thiopurine methyltransferase testing in all patients is optimal, but the testing is not routinely performed in all patients receiving mercaptopurine for ALL. It is believed the relative cost and low prevalence of TPMT deficiency is responsible for lack of testing in all patients before beginning therapy. However, TPMT testing should be strongly considered when a patient develops severe myelosuppression, and the dose should be adjusted to minimize dose reductions of concurrent anti-cancer drugs and maintain thioguanine nucleotide concentrations in agreement with the product information.

Epidermal Growth Factor Inhibitors Three anti-cancer drugs are classified as epidermal growth factor receptor (EGFR) inhibitors: erlotinib, cetuximab, and panitumumab. Erlotinib is a small molecule that inhibits the EGFR tyrosine kinase by binding to the adenosine triphosphate (ATP)-binding site of the receptor. On stimulation, two EGFRs form a homodimer and autophosphorylate each other with a molecule of ATP. Autophosphorylation leads to a conformational change that exposes protein-binding sites and subsequently initiates an intracellular signaling cascade. Erlotinib suppresses the intracellular signaling cascades by preventing autophosphorylation of the receptors. Erlotinib is used to treat nonsmall cell lung cancers and head and neck cancers, as well as other solid tumors. Tumor regression and disease stabilization occurs when it is used as second-line therapy for patients with advanced nonsmall cell lung cancer. Panitumumab and cetuximab are monoclonal antibodies that also suppress the downstream intracellular signaling cascades associated with the EGFR. These antibodies block the binding of endogenous ligands (e.g., epidermal growth factor, transforming growth factor-alpha) to the EGFR. These antibodies may also down-regulate receptor levels.

Cetuximab is used to treat both head and neck and colorectal cancers. This antibody is also used to treat locally or regionally advanced head and neck cancer in combination with radiation therapy or recurrent disease alone. It is also indicated for first-line therapy for patients with metastatic colorectal cancer alone or in combination with irinotecan, as well as recurrent disease. Panitumumab is also used to treat patients with metastatic colorectal cancer whose previous therapy with regimens containing fluoropyrimidines, irinotecan, and oxaliplatin failed. Tumor regression and disease stabilization occur in all of these settings.

EGFR Mutations and Efficacy Mutations Mutations in the EGFR coding and regulatory sequences are reported. Most polymorphisms are considered activating mutations and are associated with higher response rates to these EGFR inhibitors. Often, these mutations are associated with gene amplification. These mutations occur in 15% of patients with a diagnosis of nonsmall cell lung cancer who have a smoking history and in 50% of similar patients without a smoking history. These mutations are also common in squamous cell head and neck cancers with EGFR protein identified in most tissue specimens. Other tumors that may express these mutations include colon and breast cancers and gliomas. Acquired resistance may be associated with a secondary somatic mutation.

Erlotinib Several clinical studies have documented a positive association between EGFR somatic mutations in the kinase domain (exons 1824) and clinical response to erlotinib. The two most common mutations include an in-frame deletion in exon 19 and a missense single-nucleotide polymorphism L858R in exon 21, which account for 85% of the somatic mutations identified in patients with nonsmall cell lung cancer. These mutations are more common in patients with clinical predictors of response to erlotinib (e.g., female sex, Asian ethnicity, never-smoker, adenocarcinoma histology). One study indicates that the patients receiving erlotinib or gefitinib (another small molecule that can inhibit EGFR) with an exon 19 somatic mutation experience longer overall survival (38 months vs. 17 months) and higher response rate (73% vs. 50%) compared with patients with the exon 21 missense mutation. Epidermal growth factor receptor copy number or amplification is also associated with somatic mutations and is positively associated with a clinical response to erlotinib in several studies. In a recent study, both Kirsten retrovirusassociated DNA sequences (KRAS) wild-type and EGFR mutations (in-frame exon 19 deletion and missense exon 21 mutation) were associated with a positive response to erlotinib in patients with nonsmall cell lung cancer. Significant survival benefit from erlotinib therapy was observed for patients with wild-type KRAS and EGFR positivity by fluorescence in situ hybridization (FISH). Tumor specimens were considered FISH positive if a high degree of polysomy or amplification was noted; FISH permits semiquantification of gene copy number. The KRAS gene encodes the protein KRas, which promotes proliferation by stimulating growth factors and other intracellular signaling pathways. Kirsten retrovirusassociated DNA sequences are guanine


triphosphatases downstream of EGFR. It is estimated that KRAS mutations occur in 25% of patients with a diagnosis of lung adenocarcinoma. Earlier studies also found that KRAS mutations were predictive of resistance to erlotinib. These mutations are also associated with poor prognosis and response to cytotoxic chemotherapy. The KRAS mutations can also aid in treatment selection in patients with bronchoalveolar carcinoma or bronchoalveolar carcinoma subtype. These biomarkers have been studied extensively in patients with nonsmall cell lung cancer, but not in patients with other cancer diagnoses. However, erlotinib treatment is being examined in clinical studies for the treatment of head and neck cancers, glioma, and metastatic breast cancer. One study using archived tissue indicated that EGFR expression and localization could be associated with erlotinib response in patients with squamous cell carcinoma of the head and neck. Other studies appear to support the association between high EGFR copy number in tumor specimens and response to erlotinib in patients with head and neck cancer. Still other studies appear to indicate that these mutations are rare and not predictive of response. No distinguishing histology or associated histories appear predictive of response in patients with head and neck cancer; the disparate findings are likely because of the high frequency of EGFR mutations and KRAS wild type identified in tumor specimens of head and neck cancers. Unlike the other tumors studied to date, a specific mutation EGFRvIII appears responsible for the noted responses to erlotinib in patients with gliomas. This variant is a constitutively active genomic deletion unique to glioblastomas and leads to the persistent activation of the phosphatidylinositol 3 kinase (PI3K) signaling pathway. One study demonstrated that coexpression of this mutation and the phosphatase and tensin homolog deleted in the chromosome 10 (PTEN) tumor-suppressor protein corresponded to response to erlotinib in patients with glioblastomas. It is postulated that the PTEN protein, commonly lost in glioblastomas, maintains the link between the EGFR and the PI3K pathway in this patient population. It also appears that somatic mutations of the EGFR tyrosine kinase domain may be predictive of response to this drug in breast cancer. Erlotinib is not currently indicated for breast cancer and gliomas, but it appears to be a promising alternative therapy for some individuals with these unique EFGR mutations.

Cetuximab Cetuximab was exclusively studied in patients with metastatic colorectal cancer whose previous therapy with irinotecan and oxaliplatin failed and whose tumors stained positive for EGFR using a commercial assay. The percentage of cells staining positive for EGFR and the signal intensity were scored. However, the subpopulations of patients who respond to cetuximab remain poorly defined. Only one study indicates that patients with a higher gene copy number by FISH are more likely to respond to cetuximab, as demonstrated in studies that showed a correlation between gene copy number and response to erlotinib in patients with lung cancer. Patients with FISH-positive tumors experienced a significantly higher response rate and longer time to progression compared with FISH-negative tumors. Additional data support that tumors with wild-

type KRAS are also more likely to respond to cetuximab plus chemotherapy in chemotherapy-nave patients with metastatic colorectal cancer. The response rate increased to 59% compared with 43% for chemotherapy alone, and the risk of progression decreased to 25% compared with 43% at 1 year. It appears that EGFR gene copy number and KRAS wild type are predictive of response to cetuximab in patients with metastatic colorectal cancer. Limited studies have examined the relationship between these genes and response to cetuximab in head and neck cancer. It appears that EGFR somatic mutations are very common in this population, whereas KRAS mutations are very rare. Therefore, identifying the subpopulations more likely to respond to this antibody is unlikely, as indicated for erlotinib.

Panitumumab Panitumumab was also exclusively studied in patients with EGFR-positive tumors using the same commercial assay incorporated into the lead trials for cetuximab and recently given a diagnosis of recurrent colorectal cancer after treatment with fluoropyrimidines, oxaliplatin, and irinotecan. A recent study indicates that KRAS mutations predict lack of response to this antibody. The response rate was 0% for patients with KRAS mutation and 17% for patients with KRAS wild type. The patients with KRAS wild type also experience a longer median time to progression (12.3 months vs. 7.4 months). It appears the EGFR expression and KRAS wild type are also predictors of response to panitumumab.

Clinical Testing Epidermal growth factor receptor testing includes immunohistochemistry (IHC), FISH, and mutational analyses of the gene. Although several retrospective studies of these tests demonstrate a strong predictive value of EGFR gene expression, prospective validation is warranted to adequately determine which method will be clinically useful to predict treatment outcomes in response to erlotinib. Two studies indicate some discordance among the different methods. Epidermal growth factor receptor status is measured in tissue blocks by three methods: (1) protein expression by standardized IHC, (2) gene copy number by FISH, and (3) mutation analysis by sequencing. Substantially more tumors were positive for EGFR using IHC or FISH compared with mutation analysis. All patients positive for an activating somatic EGFR mutation experienced a complete or partial response after receiving gefitinib or erlotinib, although more than half of these tumors were IHC negative or FISH negative. In a subsequent study, a poor association between protein expression and mutation analysis was demonstrated, but a strong association between gene amplification and mutation analysis was identified. Both parameters were associated with improved response and longer time to progression and overall survival. A retrospective study also demonstrated a poor correlation between mutation analysis and IHC. A mass spectrometry assay has also been validated as part of a multi-institutional study to identify the likelihood of good or poor outcomes after treatment with erlotinib in patients with nonsmall cell lung cancer. Because the indiscriminate use of gefitinib or erlotinib in patients with nonsmall cell lung cancer produces limited


survival benefit, EGFR status should be determined before beginning therapy. In 2005, Genzyme launched the mutation assay to detect EGFR mutations in patients with nonsmall cell lung cancer. The assay combines polymerase chain reaction and gene sequencing testing technologies to detect EGFR mutations in the tyrosine kinase domain. The assay is performed on tissue biopsy material in the laboratory by trained scientists. Epidermal growth factor receptor testing is considered standard of care. In addition, KRAS testing is considered standard of care in patients with colorectal cancer before they receive either cetuximab or panitumumab. A KRAS mutation assay kit that uses a polymerase chain reactionbased test to detect the most common mutations is now available.

Tyrosine Kinase Inhibitors Chronic myeloid leukemia (CML) is characterized by the translocation of chromosomes 9 and 22, known as the Philadelphia chromosome. The fusion of these two genes causes the formation of the BCR-ABL gene, which results in an active tyrosine kinase protein. The phosphorylated protein enhances cell proliferation, differentiation, adherence, and apoptosis. Before the availability of imatinib, interferon alfa and cytarabine was considered the standard of care. Imatinib was one of the first tyrosine kinase inhibitors developed; it works by inhibiting ATP binding to BCR-ABL kinase, thereby blocking phosphorylation and downstream signaling. The efficacy of imatinib in the treatment of CML was first evaluated in a study in which 553 patients were randomized to imatinib or interferon alfa plus low-dose cytarabine. Imatinib demonstrated superior efficacy compared with interferon alfa and low-dose cytarabine in rates of major cytogenetic responses, complete cytogenetic responses, and rate of freedom from progression to the accelerated phase and blast crisis. Imatinib became the new standard of care for the treatment of CML based on its greater efficacy and improved tolerability. However, not all patients respond to therapy, and many develop resistance. Primary resistance to imatinib is defined as an initial lack of response to therapy; secondary resistance occurs when patients achieve an initial response but then develop progressive disease. Multiple mechanisms have been proposed for the development of resistance to imatinib. These include increased drug efflux by membrane transporters, increased drug binding by plasma proteins, constitutive activation of a downstream signaling pathway, amplification of the BCR-ABL gene, and mutations within the BCR-ABL kinase domain. Two newly approved agents that target the BCR-ABL fusion protein are nilotinib and dasatinib. These small molecules were rationally designed based on known mutations of the tyrosine kinase domain to overcome resistance to imatinib. Nilotinib and dasatinib also bind to the ATP-binding domain and inhibit BCR-ABL tyrosine kinase activity. Nilotinib is a more potent BCR-ABL inhibitor than imatinib, but it demonstrates activity similar to other tyrosine kinases such as Kit and platelet-derived growth factor receptor-beta. Dasatinib is also a potent inhibitor of the BCR-ABL tyrosine kinase, as well as other tyrosine kinases such as the Src family, Kit, and platelet-

derived growth factor receptor-beta. Both anti-cancer drugs are indicated to treat patients with imatinib-resistant disease and appear to demonstrate different sensitivities based on the specific mutation identified as discussed below. Subtle differences in the incidence of the more common adverse effects attributed to these drugs may be caused by the inherent differences in binding to the various receptor tyrosine kinases.

BCR-ABL Mutations The underlying mechanism of resistance to imatinib includes BCR-ABL gene amplification, BCR-ABL messenger RNA, and protein overexpression and BCR-ABL point mutations; these point mutations appear to be the most common mechanism of resistance to imatinib. The most common mutations are found in the tyrosine kinase domain, where they impair the binding of imatinib to this tyrosine kinase domain. More than 50 point mutations have been identified; the most common mutations that account for 60% to 70% of the resistance to imatinib include Gly250, Tyr253, Glu255, Thr315, Met351, and Phe359. Bone marrow or peripheral blood samples from 370 patients with CML or Philadelphia chromosomepositive ALL treated with imatinib were assessed for mutations within this kinase domain. Around 43% of the patients had at least one mutation. Mutations were more common in patients in accelerated phase or blast crisis and in patients with acquired resistance. In addition, mutations were more common in patients who had received prior interferon alfa therapy compared with patients who received imatinib as first-line therapy. The T315I mutation was more commonly identified in patients with advanced disease. Furthermore, African Americans with a diagnosis of Philadelphia chromosomepositive CML tended to have more chromosomal abnormalities compared with whites, and the median survival time was shorter in this population. However, it is not known whether African Americans have more de novo or acquired mutations in the tyrosine kinase domain. Sex or additional racial differences have not been explored.

Clinical Outcomes These mutations are de novo or acquired and lead to disease progression and decreased overall survival. One mechanism to overcome this resistance is to escalate the dose of imatinib. Previous studies have demonstrated that patients with chronic-phase CML experiencing hematologic or cytogenetic resistance to imatinib can experience a hematologic or cytogenetic response after increasing the dose of imatinib to 600800 mg/day orally. Both dasatinib and nilotinib demonstrate sensitivity to most point mutations, and clinical responses are demonstrated in clinical trials in some patients with imatinib-resistant disease. However, differences are noted in the sensitivity of these drugs to select point mutations. For example, the Phe317 mutation causes decreased sensitivity to imatinib and dasatinib but retains sensitivity to nilotinib, and the Tyr253 mutation causes resistance to imatinib and nilotinib but retains sensitivity to dasatinib. It appears that increasing doses of nilotinib or dasatinib may overcome some initial resistance to these drugs with these select mutations. Unfortunately, neither dose escalation with imatinib nor


the newer tyrosine kinase inhibitors can elicit a response in patients carrying the T315I mutation. Omacetaxine, which is currently in clinical development, may elicit a response in patients who carry the T315I mutation. This anti-cancer drug has a novel mechanism of action and induces apoptosis independent of tyrosine kinase inhibition. Omacetaxine could provide a much-needed effective therapy for carriers of the T315I mutation.

Clinical Testing Genzyme provides standardized molecular testing services as part of the CML ALLIANCE. The alliance offers a quantitative polymerase chain reaction test to measure BCR-ABL transcript concentrations in the bone marrow or peripheral blood. The test is promoted to guide therapeutic decisions regarding response to imatinib. However, this test does not detect point mutations within the tyrosine kinase domain. Tests for mutations should be considered when there is (1) failure to achieve complete hematologic response at 3 months; (2) no cytogenetic response at 6 months; (3) failure to achieve major cytogenetic response at 12 months; and (4) any sign of loss of response per the most recent clinical practice guidelines. Several laboratories offer testing to detect these mutations. Direct sequencing of the tyrosine kinase domain is completed using bone marrow aspirate or peripheral blood. Total RNA is extracted, and the first strand of complementary DNA is synthesized; the DNA is used for nested polymerase chain reactions that then undergo direct sequencing. The results usually include codon number, mutation, and abundance of the identified mutations.

Conclusion The anti-cancer drugs discussed in this chapter demonstrate substantial intersubject variability in response to therapy. The variability may range from a poor response to exaggerated toxicity in a subset of patients. It appears that polymorphisms within the drug target (e.g., EGFR, BCR-ABL) or metabolic pathway (e.g., CYP2D6, TPMT, UGT, DPD) may explain some of the variability. Studies show an association between either efficacy or tolerability and specific polymorphisms or expressions of these proteins. Clinical testing is available for some of these mutations, but an association between each known variant and outcomes has not been identified. Furthermore, ethnic differences have also not been explored for all of these mutations. However, these tests may be helpful in reducing toxicity or maximizing efficacy in some subsets and should be strongly considered in these subsets. It is anticipated that clinical testing will become more common, and that tailoring therapy based on specific molecular characteristics will become the standard of care.

Annotated Bibliography 1. Bosch TM. Pharmacogenomics of drug-metabolizing

enzymes and drug transporters in chemotherapy. Methods Mol Biol 2008;448:6376.

This review provides a thorough overview of the polymorphisms in drug-metabolizing enzymes and transporters that affect patients with cancer. Examples include TPMT, DPD, and UGT. The authors discuss the importance of associating changes in pharmacokinetics with pharmacodynamics. Furthermore, the influence of the underlying diagnosis and methodology for detecting these polymorphisms are discussed. This review highlights the need for prospective studies that show a clear relationship between clinical outcomes and these polymorphisms before clinical testing for these polymorphisms can be consistently used to tailor treatment.

2. Jin Y, Desta Z, Stearns V, Ward B, Ho H, Lee KH, et al. CYP2D6 genotype, antidepressant use, and tamoxifen metabolism during adjuvant breast cancer treatment. J Natl Cancer Inst 2005;97:309.

This study was one of the first to evaluate the influence of CYP2D6 and the use of concurrent CYP2D6 inhibitors on the metabolism of tamoxifen. This study included 80 patients with newly diagnosed breast cancer who were beginning tamoxifen. Of these patients, 24 women were taking CYP2D6 inhibitors. Genotypes CYP2D6, CYP2C9, CYP3A5, and SULT1A1 were assessed, and the concentrations of tamoxifen and its metabolites were measured after 1 and 4 months of therapy. Concentrations of endoxifen, the active metabolite of tamoxifen, were significantly lower in women heterozygous or homozygous for a variant CYP2D6 allele (p=0.003). In patients who were homozygous wild-type genotype but receiving CYP2D6 inhibitors, the plasma concentrations of endoxifen were 58% lower than those who were not taking CYP2D6 inhibitors (p=0.002). This study demonstrates a definitive relationship between genotype and phenotype and the influence of completive inhibitors on the disposition of the active metabolite.

3. Goetz MP, Rae JM, Suman VJ, Safgren SL, Ames MM, Visscher DW, et al. Pharmacogenetics of tamoxifen biotransformation is associated with clinical outcomes of efficacy and hot flashes. J Clin Oncol 2005;23:93128.

This was the first study to associate the CYP2D6 genotype with disease outcome. The study assessed 223 paraffin-embedded tumor samples from patients who had received 5 years of adjuvant tamoxifen therapy. Alleles of CYP2D6 (*4 and *6) and CYP3A5 (*3) were evaluated. The primary outcomes assessed were relapse-free time, disease-free survival, and overall survival. Women homozygous for the CYP2D6*4 allele had a shorter relapse-free time (p=0.023) and a lower disease-free survival (p=0.012) compared with women heterozygous or homozygous for the wild-type allele. Overall survival was not statistically different (p=0.169). Genotypes identified in DNA extracted from tumor samples and buccal swabs were compared; the concordance between buccal swabs and tumor samples was 100% for CYP2D6*4 (15 women) and CYP3A5*3 (13 women). These additional data indicate that buccal swabs may be used in place of paraffin-embedded tumor samples to predict clinical outcomes.

4. Hoskins JM, Goldberg RM, Qu P, Ibrahim JG, McLeod HL. UGT1A1*28 genotype and irinotecan-induced neutropenia: dose matters. J Natl Cancer Inst 2007;99:12905.

This meta-analysis included nine studies with a total of 821 patients and assessed the association between irinotecan dose and severe hematologic toxicity in patients homozygous for the variant allele UGT1A1*28. This study was the first to demonstrate that toxicity associated with this variant allele is a


function of the dose. The doses were defined as low (less than 150 mg/m2), medium (150250 mg/m2), and high (more than 250 mg/m2) based on the most common doses administered to patients. Patients who were carriers of this variant allele and received higher doses of irinotecan were more likely to develop severe toxicity compared with patients with the same genotype receiving lower doses. When patients with the UGT1A1*28/*28 genotype were compared with patients who were UGT1A1*1/*1 or UGT1A1*1/*28, the risk of toxicity in the first group was greater at medium doses (odds ratio [OR] = 3.22, 95% confidence interval [CI] = 1.526.81, p=0.008) and high doses (OR = 27.8, 95% CI = 4195, p=0.005). At lower doses, the risk was not statistically different (OR = 1.8, 95% CI = 0.378.84, p=0.41). This study suggests that genotyping should only be conducted in patients receiving higher doses (i.e., more than 150 mg/m2) of irinotecan.

5. Yen JL, McLeod HL. Should DPD analysis be required prior to prescribing fluoropyrimidines? Eur J Cancer 2007;43:10116.

This concise review discusses the metabolism of fluorouracil and the relationship between DPD and clinical outcomes. As discussed in the text, treatment of patients with fluorouracil-based chemotherapy can be accompanied by severe and sometimes lethal toxicity. Dihydropyrimidine dehydrogenase plays a pivotal role in the metabolism of fluorouracil, and DPD deficiency has been recognized as a risk factor for the development of severe toxicity and clinical outcomes. The various methods to measure DPD deficiency are thoroughly discussed, including potential limitations and eventual clinical application. Screening of patients for the presence of a DPD deficiency before treatment is not routinely performed, but the authors conclude that screening for DPD deficiency may become standard of care as progress continues regarding the understanding of the molecular basis of the deficiency and the development of clinical assays.

6. Crews KR. Individualizing chemotherapeutic treatment of colorectal cancer. Am J Health Syst Pharm 2006;63(Suppl 2):S12S17.

This review summarizes previously published reports regarding polymorphisms in genes that encode drug-metabolizing enzymes, receptors of drugs used to treat colorectal cancer, and the association between these polymorphisms and clinical outcomes. The article focuses on the relationship between fluorouracil and DPD deficiency, irinotecan and UGT tandem repeats, and cetuximab and EGFR mutations. The article also discusses tailoring chemotherapy based on traditional risk factors in addition to genetic polymorphisms. Assays used for genotypic testing, protein expression, and metabolic capacity are described. Overall, it provides a concise review of the data available for patients with colorectal cancer and places the data in context regarding clinical testing and therapeutic decisions.

7. McLeod HL, Coulthard S, Thomas AE, Pritchard SC, King DJ, Richards SM, et al. Analysis of thiopurine methyltransferase variant alleles in childhood acute lymphoblastic leukaemia. Br J Haematol 1999;105:696700.

The initial clinical studies that found an association between TPMT deficiency and clinical outcomes in children with ALL were conducted more than 10 years ago. This study included 147 children with acute leukemia, and 11% were identified as carriers of the most common variant allele. Hematologic toxicity prevented patients from receiving maintenance therapy on a mean of 11% of the weeks of

maintenance therapy and demonstrated a trend toward an association between genotype and dose interruptions. The number of weeks of maintenance therapy omitted for myelosuppression was similar between homozygotes and heterozygotes for the wild-type genotype but markedly lower for the patient identified as a homozygote for variant genotype. This is one of the first studies to demonstrate the impact of TPMT genotype on treatment.

8. Relling MV, Hancock ML, Rivera GK, Sandlund JT, Ribeiro RC, Krynetski EY, et al. Mercaptopurine therapy intolerance and heterozygosity at the TPMT gene locus. J Natl Cancer Inst 1999;91:20018.

This article describes the first prospective study that characterized the relationship between TPMT alleles and deficiency. One hundred eighty patients received oral mercaptopurine plus intravenous methotrexate as part of the maintenance therapy for ALL. Thioguanine nucleotide levels and TPMT activity were measured in red blood cells. The activity of TPMT was used to identify the patients as homozygous wild type, heterozygous, or homozygous variant. The exact dose and reasons for deviations were recorded for each dose. Mean thioguanine nucleotide values were inversely related to TPMT activity (p


p=0.35). A multivariate analysis, however, indicated only that EGFR FISHpositive status was prognostic for worse survival in untreated patients (p=0.025) and predictive of a greater survival benefit from erlotinib (p=0.005). This study was one of the first to indicate that EGFR mutation status alone may not be adequate to predict response to these small molecules.

11. Baccarani M, Saglio G, Goldman J, Hochhaus A, Simonsson B, Appelbaum F, et al. Evolving concepts in the management of chronic myeloid leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet. Blood 2006;108:180920.

This article provides recommendations on the treatment of patients with early chronic-phase CML. The most current literature regarding the use of interferon alfa, transplantation, and imatinib is reviewed. The use of imatinib and dosage increases in patients who do not respond to therapy is also discussed. In addition, the topic of resistance to therapy and mutations is addressed. A list of the common mutations and the IC50 (inhibitory concentration of 50%) values for imatinib are provided. Definitions of response and monitoring, as well as treatment recommendations, are provided. This review article provides a concise overview of the treatment of CML and a thorough description of the use of imatinib in patients who develop resistance.

12. OHare T, Eide CA, Deininger MW. Bcr-Abl kinase domain mutations, drug resistance, and the road to a cure for chronic myeloid leukemia. Blood 2007;110:22429.

The authors review the most common mutations identified in the BCR-ABL kinase domain and discuss how to treat patients with these mutations. Several questions, including how do point mutations cause resistance, when do mutations occur, and what factors are driving resistance in patients, are answered in this review. A colorful table identifies which mutations demonstrate sensitivity to each of these small molecules, and a discussion regarding the impact of nilotinib and dasatinib on the emergence of these mutations is addressed. This well-organized review helps readers address concerns regarding the use of these inhibitors in patients with CML.


41. A specific metabolic enzyme catalyzes the rate-limiting step in the elimination of a new anti-cancer drug. The maximal plasma concentrations of the parent drug vary about 25-fold in patients. It is discovered that about 10% of patients develop substantial toxicity and can no longer tolerate the drug. In this subset, parent drug concentrations are significantly higher than anticipated. No drug-drug interactions can be identified in these patients. Molecular profiling indicates that messenger RNA and concentrations of the metabolic enzyme are substantially reduced. Furthermore, a base substitution in the ligand-binding domain that leads to an amino acid substitution is identified. Which one of the following types of mutations is the most likely type of mutation identified?

A. Single nucleotide polymorphism.B. Tandem repeat.C. Copy number.D. Insertion.

42. A mutation in a ligand-binding domain of a drug-metabolizing enzyme corresponds to a decrease in the active metabolite and corresponding increases in the parent drug in an animal model for a new anti-cancer drug. Clinical studies subsequently confirmed the relationship between the mutation and the pharmacokinetics in humans, but Phase II studies did not identify an association between the changes in pharmacokinetics and increases in the incidence of grade 3 or 4 toxicity or response. Genetic testing in these studies included a relatively simple polymerase chain reaction, followed by biallelic determination, which may be performed in most laboratories. However, this mutation occurs in only about 1% of whites, and its frequency in African Americans, Hispanics, and Asians is not known yet. Which one of the following characteristics of this mutation is the best support for clinical testing?A. Simple, available assay.B. Associated with toxicity.C. Ethnic variability.D. High prevalence.

43. A 45-year-old white woman recently completed adjuvant chemotherapy for early-stage breast cancer. She is premenopausal, and the tumor is hormone receptor positive. Tamoxifen 20 mg/day given orally was initiated immediately after the completion of chemotherapy. She started taking paroxetine for depression. Within 1 year, the breast cancer recurs. Which one of the following is the most plausible explanation for her recurrence?A. A drug-drug interaction.B. She is a cytochrome P450 (CYP) 2D6 extensive

metabolizer.C. Nonadherence.D. Premenopausal status.

44. A 37-year-old African American woman with early-stage breast cancer recently completed adjuvant chemotherapy after a lumpectomy. The woman is premenopausal, and the tumor was hormone receptor positive. Her medical history notes she does not respond to codeine, and she experiences substantial adverse effects after taking paroxetine for depression. Which one of the following is the best endocrine therapy for this woman at this time?A. Oophorectomy.B. Tamoxifen.C. Anastrozole.D. Goserelin.

45. A 53-year-old premenopausal woman recently given a diagnosis of breast cancer just completed adjuvant chemotherapy. She plans to take tamoxifen because her tumor is hormone receptor positive. She is considered a poor responder to codeine and hydrocodone, two drugs that require metabolism by CYP2D6 to the active compound. Which one of the following outcomes is most likely based on her suspected CYP2D6 metabolic status?A. Increased disease-free survival.B. Increased hot flashes.C. Decreased overall survival.D. Decreased relapse-free survival.

46. A 65-year-old white man with a diagnosis of locally advanced colon cancer received combination chemotherapy with oxaliplatin and fluorouracil, but his disease progressed after three cycles. Subsequently, he will receive combination chemotherapy with fluorouracil, irinotecan, and leucovorin every 2 weeks. His laboratory values indicate he is unable to conjugate bilirubin effectively. Which one of the following is the most important action before beginning therapy with irinotecan?A. Provide filgrastim to minimize myelosuppression.B. Start loperamide to minimize diarrhea.C. Complete clinical testing for uridine diphosphate-

glucuronosyltransferase (UGT)1A1*28.D. Reduce the irinotecan dose to 80% of the usual

starting dose.

47. A 52-year-old Hispanic woman undergoes clinical testing for UGT1A1 polymorphism after developing severe neutropenia (absolute neutrophil count less than 500 cells/mm3) after receiving one dose of irinotecan. The woman is receiving irinotecan in combination with fluorouracil for first-line treatment of metastatic colorectal cancer. The test indicates the woman is homozygous for the variant allele. Which one of the following alternatives is best for this woman at this time?A. Discontinue irinotecan; begin alternative therapy.

Self-Assessment Questions


B. Decrease the irinotecan dose to 20% of the usual dose.

C. Administer filgrastim to increase white blood cell count.

D. Reinitiate irinotecan at the current dose once the white blood cell count increases.

48. A 50-year-old white man with a diagnosis of metastatic colorectal cancer recently underwent first-line chemotherapy, which failed. He is to begin second-line therapy with fluorouracil, leucovorin, and irinotecan every 2 weeks. The patient expresses normal dihydropyrimidine dehydrogenase (DPD) activity and is a carrier for the variant UGT1A1*28 allele. Which one of the following most accurately describes the potential impact of his metabolic capacity on his response to or tolerance of this combination anti-cancer drug regimen?A. More severe bone marrow suppression with

fluorouracil.B. Improved clinical response to fluorouracil.C. More severe diarrhea and myelosuppression with

irinotecan.D. A good response to irinotecan with prolonged

exposure to SN-38.

49. A patient developed grade 3 or 4 neutropenia and diarrhea after receiving a single irinotecan dose of 300 mg/m2 over 90 minutes. The patient is determined not to be a carrier of the variant allele UGT1A1*28 after a commercial assay is completed to identify the genotype for this enzyme. Serum creatinine is 1.1 mg/dL, total bilirubin is 1.3 mg/dL, and transaminases are 1.2 times the upper limit of normal. Which one of the following is the most plausible explanation for the development of severe toxicity in this patient?A. Reduced hepatic clearance of irinotecan.B. Reduced urinary clearance of irinotecan.C. Alternative mutations in the UGT1A1 gene.D. Decreased carboxylesterase activity.

50. A 68-year-old white man was given a diagnosis of locally advanced pancreatic cancer. When he did not respond to three cycles of gemcitabine, he opted to start bolus fluorouracil with radiation therapy. If it were available, which one of the following options would be the best reason for offering clinical testing of DPD in this man?A. DPD activity is lower in African Americans.B. DPD activity is lower in women than men.C. DPD deficiency corresponds with increased

toxicity.D. DPD deficiency corresponds with improved

outcomes.

51. A 63-year-old Hispanic woman begins capecitabine plus lapatinib for metastatic breast cancer that had progressed on previous systemic therapy. The woman develops grade 3 neutropenia after starting therapy. She takes capecitabine with food and lapatinib without

food. Which one of the following is the most likely explanation for this toxicity?A. DPD deficiency.B. Decreased carboxylesterase activity.C. Drug-food interaction.D. Drug-drug interaction.

52. A 3-year-old Asian Indian boy with acute lymphoblastic leukemia (ALL) completed induction and consolidation therapy with myelosuppressive chemotherapy and achieved complete remission. The boy then began maintenance therapy with mercaptopurine, vincristine, methotrexate, and prednisone. After one cycle, he developed grade 3 neutropenia. Which one of the following statements is the best reason to determine thiopurine methyltransferase (TPMT) genotype in this patient?A. Severe myelosuppression developed with the first

cycle of chemotherapy.B. Dose interruptions correspond with poorer

response.C. TPMT mutations have not been identified in Asian

Indians.D. Doses of other myelosuppressive drugs may be

modified.

53. A 7-year-old girl with ALL is starting maintenance chemotherapy with oral methotrexate 20 mg/m2/day and oral mercaptopurine 75 mg/m2/day. She is 122 cm (48 inches) tall and weighs 11 kg (25 lb). Clinical testing for TPMT shows that she is a heterozygous for a variant allele associated with reduced metabolism and increased grade 3 and 4 myelosuppression. Which one of the following mercaptopurine doses is best for this girl at this time?A. 100 mg.B. 65 mg.C. 25 mgD. 10 mg.

54. A 4-year-old boy with a diagnosis of ALL is prescribed a complex chemotherapy regimen that includes maintenance therapy with mercaptopurine. Basing chemotherapy decisions on TPMT genotype is most likely to result in which one of the following?A. Reduced risk of drug-induced neutropenia.B. Shortened course of chemotherapy.C. Elimination of the need for hematologic

monitoring.D. Reduced need for a multiple-drug chemotherapy

regimen.

Questions 55 and 56 pertain to the following case.D.B. is a 70-year-old white man with a recent diagnosis of Philadelphia chromosomepositive chronic myeloid leukemia in the chronic phase. He is not a candidate for a hematopoietic stem cell transplantation because of advanced age. The patient was initiated on imatinib 400 mg/day given orally. After 3 months, the patient achieved a hematologic response, and at 6 months, the patient achieved a cytogenetic


response. However, a bone marrow biopsy at 12 months showed accelerated disease.

55. Which one of the following factors places D.B. at increased risk of a tyrosine kinase domain mutation?A. Age.B. Race.C. Disease phase.D. Prior therapy.

56. A mutational analysis demonstrated that D.B. has a T315I mutation. Which one of the following is the best recommendation for therapy for D.B. at this time?A. Imatinib 600 mg/day orally.B. Dasatinib 100 mg/day orally.C. Nilotinib 200 mg/day orally.D. Participation in a clinical trial.

57. A 72-year-old white woman received a diagnosis of stage IIIB adenocarcinoma of the lung. She also has a 104 pack-year history of smoking. Chemotherapy with paclitaxel plus carboplatin was administered for four cycles, but the patient experienced disease progression. She plans to begin therapy with erlotinib. Which one of the following patient and/or tumor characteristics is most likely to be associated with an increased response to erlotinib in this patient?A. Smoking history.B. Tumor subtype.C. Age.D. Race.

58. A 55-year-old Asian woman has a diagnosis of large cell lung cancer. Her medical history includes no illicit drugs or alcohol abuse, but she smoked about one-half pack of cigarettes per day for 30 years. She quit smoking about 7 years ago. Mutational analysis indicates epidermal growth factor receptor (EGFR) overexpression and Kirsten retrovirusassociated DNA sequences (KRAS) variant. Which one of the following characteristics is associated with a greater likelihood of response to erlotinib in this woman?A. Race.B. Sex.C. Tumor subtype.D. KRAS mutation.

59. The mutation analysis for EGFR domain has been examined in multiple studies. The tumor tissue from an individual patient shows immunohistochemistry (IHC) positive, fluorescence in situ hybridization (FISH) negative, and EGFR altered sequence. Which one of the following tests is the best predictor of response to erlotinib?A. Western blot.B. IHC.C. FISH.D. Mutation analysis.

60. A 55-year-old man receives a diagnosis of recurrent colorectal cancer after receiving oxaliplatin with fluorouracil as first-line therapy and irinotecan plus fluorouracil as second-line therapy. He will begin taking cetuximab alone. Which one of the following assays should be conducted as part of standard of care before beginning therapy?A. KRAS.B. EGFR.C. UGT.D. DPD.

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