Role of UDP-Glucuronosyltransferase 1A1 in the Metabolism and Pharmacokinetics of Silymarin Flavonolignans in Patients with HCV and NAFLD.

Silymarin is the most commonly used herbal medicine by patients with chronic liver disease. Silymarin flavonolignans undergo rapid first-pass metabolism primarily by glucuronidation. The aims of this investigation were: (1) to determine the association of UGT1A1*28 polymorphism with the area under the plasma concentration-time curves (AUCs) for silybin A (SA) and silybin B (SB); (2) to evaluate the effect of UGT1A1*28 polymorphism on the profile of flavonolignan glucuronide conjugates found in the plasma; and (3) to investigate the role of UGT1A1 enzyme kinetics on the pharmacokinetics of SA and SB. AUCs and metabolic ratios for thirty-three patients with chronic liver disease administered oral doses of silymarin were compared between different UGT1A1*28 genotypes. The AUCs, metabolic ratios, and the profiles of major SA and SB glucuronides did not differ significantly among the three UGT1A1 genotypes. In contrast, an increase in the proportion of sulfated flavonolignan conjugates in plasma was observed in subjects with UGT1A1*28/*28 genotype compared to subjects carrying wild type alleles. Differences in SA and SB in vitro intrinsic clearance estimates for UGTIA1 correlated inversely with SA and SB exposures observed in vivo indicating a major role for UGT1A1 in silymarin metabolism. In addition, a significant difference in the metabolic ratio observed between patients with NAFLD and HCV suggests that any effect of UGT1A1 polymorphism may be obscured by a greater effect of liver disease on the pharmacokinetics of silymarin. Taken together, these results suggest the presence of the UGT1A1*28 allele does not contribute significantly to a large inter-subject variability in the pharmacokinetics of silybin A and silybin B which may obscure the ability to detect beneficial effects of silymarin in patients with liver disease.

Fixed-dose capecitabine is feasible: results from a pharmacokinetic and pharmacogenetic study in metastatic breast cancer.

The pro-drug capecitabine is approved for treatment of anthracycline- and paclitaxel-resistant metastatic breast cancer. However, toxicity and large interpatient pharmacokinetic variability occur despite body surface area (BSA)-dosing. We hypothesized that a fixed-dose schedule would simplify dosing and provide an effective and safe alternative to BSA-based dosing. We conducted an open label, single-arm, two-stage study of oral capecitabine with fixed starting dose (3,000 mg total daily dose in two divided doses × 14 days q21 days) in patients with metastatic breast cancer. We correlated pharmacodynamic endpoints [e.g., efficacy (response) per RECIST and toxicity], adherence and pharmacokinetics/pharmacogenetics. Sample size of 45 patients was required to detect a 25 % response rate from null response rate of 10 % using a Simon two-stage design. Twenty-six patients were enrolled in the first-stage and 21 were evaluable after a median of four cycles of capecitabine. Two thirds of patients received either the same dose or a dose 500 mg lower than what would have been administered with a commonly used 2,000 mg/m(2) BSA-dosing schedule. Eight patients had stable disease but progressed after a median of seven cycles. Despite a clinical benefit rate of 19 %, no RECIST responses were observed following the first stage and the study was closed. Dose-reductions were required for grade 2 hand-foot syndrome (28 %) and vomiting (5 %). Adherence was similar when using both patient-reported and Medication Event Monitoring System methods. High interpatient variability was observed for capecitabine and metabolite pharmacokinetics, but was not attributed to observed pharmacogenetic or BSA differences. Single agent activity of capecitabine was modest in our patients with estrogen receptor-positive or -negative metastatic breast cancer and comparable to recent studies. BSA was not the main source of pharmacokinetic variability. Fixed-dose capecitabine is feasible, and simplifies dosing.

Galactose-α-1,3-galactose-specific IgE is associated with anaphylaxis but not asthma.

RATIONALE: IgE antibodies to the mammalian oligosaccharide galactose-α-1,3-galactose (α-gal) are common in the southeastern United States. These antibodies, which are induced by ectoparasitic ticks, can give rise to positive skin tests or serum assays with cat extract. OBJECTIVES: To evaluate the relationship between IgE antibodies to α-gal and asthma, and compare this with the relationship between asthma and IgE antibodies to Fel d 1 and other protein allergens. METHODS: Patients being investigated for recurrent anaphylaxis, angioedema, or acute urticaria underwent spirometry, exhaled nitric oxide, questionnaires, and serum IgE antibody assays. The results were compared with control subjects and cohorts from the emergency department in Virginia (n = 130), northern Sweden (n = 963), and rural Kenya (n = 131). MEASUREMENTS AND MAIN RESULTS: Patients in Virginia with high-titer IgE antibodies to α-gal had normal lung function, low levels of exhaled nitric oxide, and low prevalence of asthma symptoms. Among patients in the emergency department and children in Kenya, there was no association between IgE antibodies to α-gal and asthma (odds ratios, 1.04 and 0.75, respectively). In Sweden, IgE antibodies to cat were closely correlated with IgE antibodies to Fel d 1 (r = 0.83) and to asthma (P < 0.001). CONCLUSIONS: These results provide a model of an ectoparasite-induced specific IgE response that can increase total serum IgE without creating a risk for asthma, and further evidence that the main allergens that are causally related to asthma are those that are inhaled.

Assessing the utility of whole genome amplified DNA as a template for DMET Plus array.

BACKGROUND: Large amounts of high quality DNA are typically required for high-throughput genotyping arrays but sometimes study participant DNA is in limited supply. Multiple displacement amplification (MDA)-based whole genome amplification is an in vitro technique that permits the genetic analysis of limited amounts of high molecular weight genomic DNA (gDNA). METHODS: The performance of MDA-whole genome amplified DNA (wgaDNA) as a template for DMET Plus (Affymetrix) was assessed. wgaDNA was generated from gDNA from three HapMap CEU cell lines and 11 breast cancer patients. One HapMap sample and three patient samples were randomly selected for replication to assess reproducibility. Accuracy was assessed by comparing the wgaDNA genotypes with gDNA genotypes. The kappa (κ) statistic was used to measure genotype concordance between paired gDNA-wgaDNA and wgaDNA-wgaDNA samples. Copy number variants (CNV) were not included in concordance analysis in this study. RESULTS: A good genotype call rate of 98.8%±1.06% (mean±standard deviation, 1931 markers) was observed for all 18 wgaDNA samples with three samples having call rates lower than 98%. High genotype concordance rates were observed between four HapMap wgaDNA-gDNA pairs (98.5%, κ=0.9817, p<0.0001, 1931 markers) and 14 patient wgaDNA-gDNA pairs (100%, κ=1.00, p<0.0001, 19 markers among CYP2D6 and CYP2C19). Excellent genotype concordance was also observed between four independently amplified duplicate samples (98.0%, κ=0.9745; p<0.0001, 1931 markers). CONCLUSIONS: MDA-produced wgaDNA provides accurate and reproducible genotypes with the DMET Plus array and is therefore a suitable template for this targeted pharmacogenetic genotyping array.

Population pharmacokinetic modeling of the association between 63396C->T pregnane X receptor polymorphism and unboosted atazanavir clearance.

Atazanavir (ATV) plasma concentrations are influenced by CYP3A4 and ABCB1, which are regulated by the pregnane X receptor (PXR; NR1I2). PXR expression is correlated with CYP3A4 in liver in the absence of enzyme inducers. The PXR single nucleotide polymorphism (SNP) 63396C→T (rs2472677) alters PXR expression and CYP3A4 activity in vitro, and we previously showed an association of this polymorphism with unboosted ATV plasma concentrations. The aim of this study was to develop a population pharmacokinetic analysis to quantify the impact of 63396C→T and diurnal variation on ATV clearance. A population analysis was performed with 323 plasma samples from 182 randomly selected patients receiving unboosted ATV. Two hundred fifty-nine of the blood samples were collected at random time points, and 11 patients had a full concentration-time profile at steady state. Nonlinear mixed effects modeling was applied to explore the effects of PXR 63396C→T, patient demographics, and diurnal variation. A one-compartment model with first-order absorption and lag time best described the data. Population clearance was 19.7 liters/h with interpatient variability or coefficient of variation (CV) of 21.5%. Homozygosity for the T allele for PXR 63396 was associated with a 17.0% higher clearance that was statistically significant. Evening dosing was associated with 34% higher bioavailability than morning dosing. Patient demographic factors had no effect on ATV clearance. These data show an association of PXR 63396C→T and diurnal variation on unboosted ATV clearance. The association is likely to be mediated through an effect on hepatic PXR expression and therefore expression of its target genes (e.g., CYP3A4, SLCO1B1, and ABCB1), which are known to be involved in ATV clearance.

Thymidylate synthase and methylenetetrahydrofolate reductase gene polymorphisms and toxicity to capecitabine in advanced colorectal cancer patients.

PURPOSE: To evaluate the effect of thymidylate synthase (TYMS) and methylenetetrahydrofolate reductase (MTHFR) genotypes on toxicity in patients treated with capecitabine for advanced colorectal cancer and to determine the effect of these polymorphisms on the pretreatment levels of serum folate and plasma homocysteine. EXPERIMENTAL DESIGN: Fifty-four patients with a diagnosis of metastatic colorectal cancer were treated with fixed-dose capecitabine. Germ line DNA from patients was genotyped for TYMS TSER, TSER*3G>C, and 3'-untranslated 6 bp insertion/deletion (3' untranslated region insertion/deletion), and MTHFR c.677C>T and c.1298A>C using PCRs and RFLP. Toxicity was graded by National Cancer Institute Common Toxicity Criteria version 2.0. Response was assessed by Response Evaluation Criteria in Solid Tumors. RESULTS: MTHFR c.677C>T and c.1298A>C genotypes and diplotypes predicted for grade 2/3 toxicities, whereas the TYMS genotypes had no influence. MTHFR c.677 genotype tended to predict overall survival (P = 0.08). MTHFR c.677 influenced pretreatment homocysteine (P < 0.05) and serum folate levels (P < 0.05). Multivariate analysis suggests that MTHFR c.1298 is an independent predictor of toxicity. CONCLUSIONS: This study suggests that common genetic variation in MTHFR but not TYMS may be useful for predicting toxicity from capecitabine in patients with advanced colorectal cancer. In addition, MTHFR single nucleotide polymorphisms predicted serum folate and plasma homocysteine levels, and, combined, these factors may be important predictors of capecitabine-induced toxicity.

Irinotecan pharmacogenetics: influence of pharmacodynamic genes.

PURPOSE: Irinotecan is an important drug for the treatment of solid tumors. Although genes involved in irinotecan pharmacokinetics have been shown to influence toxicity, there are no data on pharmacodynamic genes. CDC45L, NFKB1, PARP1, TDP1, and XRCC1 have been shown to influence the cytotoxic action of camptothecins, including irinotecan. Polymorphisms in the drug target of camptothecins, topoisomerase I (TOP1), and downstream effectors may influence patient outcomes to irinotecan therapy. We undertook a retrospective candidate gene haplotype association study to investigate this hypothesis. EXPERIMENTAL DESIGN: Haplotype compositions of six candidate genes were constructed in European (n = 93), East Asian (n = 94), and West African (n = 95) populations. Haplotype-tagging single nucleotide polymorphisms (htSNP) were selected based on genealogic relationships between haplotypes. DNA samples from 107 European, advanced colorectal cancer patients treated with irinotecan-based regimens were genotyped for htSNPs as well as three coding region SNPs. Associations between genetic variants and toxicity (grade 3/4 diarrhea and neutropenia) or efficacy (objective response) were assessed. RESULTS: TOP1 and TDP1 htSNPs were related to grade 3/4 neutropenia (P = 0.04) and response (P = 0.04), respectively. Patients homozygous for an XRCC1 haplotype (GGCC-G) were more likely to show an objective response to therapy than other patients (83% versus 30%; P = 0.02). This effect was also seen in a multivariate analysis (odds ratio, 11.9; P = 0.04). No genetic variants were associated with diarrhea. CONCLUSIONS: This is the first comprehensive pharmacogenetic investigation of irinotecan pharmacodynamic factors, and our findings suggest that genetic variation in the pharmacodynamic genes may influence the efficacy of irinotecan-containing therapies in advanced colorectal cancer patients.

Hepatic technetium Tc 99m-labeled sestamibi elimination rate and ABCB1 (MDR1) genotype as indicators of ABCB1 (P-glycoprotein) activity in patients with cancer.

BACKGROUND AND OBJECTIVE: The adenosine triphosphate-binding cassette transporter ABCB1 (P-glycoprotein) mediates terminal excretion of many chemotherapeutic agents, and variable ABCB1 activity may be an important contributor to interpatient variability in the clearance of chemotherapeutic agents. Our objective was to determine the elimination constant (kH) for hepatic elimination of technetium Tc 99m-labeled sestamibi (99mTc-MIBI) in patients with cancer and to compare this putative indicator of ABCB1 phenotype with clinical features and common ABCB1 genetic variants. METHODS: 99mTc-MIBI kH was determined from the time-dependent elimination profile of 99mTc-MIBI over a 90-minute hepatic scanning period in 66 patients with cancer. Single nucleotide polymorphisms (SNPs) in ABCB1 exons 12 (C1236T), 21 (G2677T/A), and 26 (C3435T) were documented by polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: There was a 12-fold variation in 99mTc-MIBI kH across the cohort, which was not correlated with sex, age, conventional liver function test results, previous chemotherapy treatment, or history of liver metastasis. Mean 99mTc-MIBI kH was significantly reduced in patients with SNPs in exons 21 and 26 such that mean 99mTc-MIBI kH was 1.90 times (95% confidence interval, 1.14-2.66; P = .02) and 2.21 times (95% confidence interval, 1.47-2.97; P < .01) higher in subjects homozygous for the wild-type alleles than in those homozygous for these SNPs, respectively. CONCLUSION: Hepatic elimination of 99mTc-MIBI is a potential in vivo probe of hepatic ABCB1 activity that is significantly associated with the presence of common SNPs in ABCB1. 99mTc-MIBI hepatic scanning may provide a useful pretreatment indicator of ABCB1-mediated drug clearance in cancer patients.

Accuracy of SNPs to predict risk of HLA alleles associated with drug-induced hypersensitivity events across racial groups.

AIM: To evaluate the potential usefulness of selected SNPs to predict specific HLA alleles that are associated with drug-induced hypersensitivity reactions (HSR) in different ethnic groups. METHODS & RESULTS: Five specific HLA alleles known to predict HSR were tagged by seven SNPs (rs1061235-HLA-A*31:01; rs2395029-HLA-B*57:01; rs3909184-HLA-B*15:02; rs9469003-HLA-B*58:01; rs3117583-HLA-B*58:01; rs9270986-HLA-DQA1*01:02 and rs3129900-HLA-DQA1*01:02). DNA from 24 African-Americans, 56 Asian, 44 Caucasians and 36 Hispanics of known high resolution HLA-A, B and DQA1 status were genotyped for tagSNPs using TaqMan. Sensitivity and specificity were considered the primary end points and were 100% across the four populations for rs2395029-HLA-B*57:01. SNP prediction of HLA-A*31:01 had 100% sensitivity and 84% specificity. CONCLUSION: This study demonstrates the utility of SNP tagging as a 'real time' approach to predict or exclude the presence of specific HLA alleles of known importance to HSR across diverse ethnic groups. Original submitted 24 April 2014; Revision submitted 2 April 2015.

Results of a pilot multicenter genotype-based randomized placebo-controlled trial of propranolol to reduce pain after major thermal burn injury.

BACKGROUND: Results of previous studies suggest that ß-adrenoreceptor activation may augment pain, and that ß-adrenoreceptor antagonists may be effective in reducing pain, particularly in individuals not homozygous for the catechol-O-methyltransferase (COMT) high-activity haplotype. MATERIALS AND METHODS: Consenting patients admitted for thermal burn injury at participating burn centers were genotyped; those who were not high-activity COMT homozygotes were randomized to propranolol 240 mg/d or placebo. Primary outcomes were study feasibility (consent rate, protocol completion rate) and pain scores on study days 5 to 19. Secondary outcomes assessed pain and posttraumatic stress disorder symptoms 6 weeks postinjury. RESULTS: Seventy-seven percent (61/79) of eligible patients were consented and genotyped, and 77% (47/61) were genotype eligible and randomized. Ninety-one percent (43/47) tolerated study drug and completed primary outcome assessments. In intention-to-treat and per-protocol analyses, patients randomized to propranolol had worse pain scores on study days 5 to 19. CONCLUSIONS: Genotype-specific pain medication interventions are feasible in hospitalized burn patients. Propranolol is unlikely to be a useful analgesic during the first few weeks after burn injury.

Effect of short-term morphine exposure on P-glycoprotein expression and activity in cancer cell lines.

Multidrug resistance (MDR) is a common problem in various types of cancer. One important factor in the development of MDR is overexpression of P-glycoprotein, encoded by the MDR1 gene. Morphine is the opioid of choice for moderate to severe cancer pain, and is a substrate of P-glycoprotein. Recently, morphine has been shown to induce P-glycoprotein expression in the rat brain. Using Western blot analysis and cytotoxicity assays respectively, we have investigated the effects of short-term (72 h) morphine treatment on P-glycoprotein expression in a panel of human cancer cell lines, and its effects on cellular resistance to the known P-glycoprotein substrates, vinblastine and colchicine. The effect of morphine on P-glycoprotein expression and activity in the mouse fibroblast NIH-3T3 cell was assessed to establish whether morphine effects are species specific. Short-term exposure to morphine did not result in any significant differences in P-glycoprotein expression or activity in any cancer cell lines. Morphine pre-treatment resulted in a moderate but significant increase in sensitivity of NIH-3T3 cells to vinblastine, but not colchicine. This study suggests that morphine effects may be cell-type specific. Importantly, however, it appears that short-term morphine treatment does not affect the MDR phenotype of tumour cells.

Association of eleven common, low-penetrance colorectal cancer susceptibility genetic variants at six risk loci with clinical outcome.

BACKGROUND: Low-penetrance genetic variants have been increasingly recognized to influence the risk of tumor development. Risk variants for colorectal cancer (CRC) have been mapped to chromosome positions 8q23.3, 8q24, 9p24.1, 10p14, 11q23, 14q22.2, 15q13, 16q22.1, 18q21, 19q13.1 and 20p12.3. In particular, the 8q24 single nucleotide polymorphism (SNP), rs6983267, has reproducibly been associated with the risk of developing CRC. As the CRC risk SNPs may also influence disease outcome, thus in this study, we evaluated whether they influence patient survival. METHODOLOGY/PRINCIPAL FINDINGS: DNA samples from 583 CRC patients enrolled in the prospective, North Carolina Cancer Care Outcomes Research and Surveillance Consortium Study (NC CanCORS) were genotyped for 11 CRC susceptibility SNPs at 6 CRC risk loci. Relationships between genotypes and patient survival were examined using Cox regression analysis. In multivariate analysis, patients homozygous for the CRC risk allele of rs7013278 or rs7014346 (both at 8 q24) were only nominally significant for poorer overall survival compared to patients homozygous for the protective allele (hazard ratio = 2.20 and 1.96, respectively; P<0.05). None of these associations, however, remained statistically significant after correction for multiple testing. The other nine susceptibility SNPs tested were not significantly associated with survival. CONCLUSIONS/SIGNIFICANCE: We did not find evidence of association of CRC risk variants with patient survival.

Catechol-O-methyltransferase genotype predicts pain severity in hospitalized burn patients.

Increasing evidence suggests that stress system activation after burn injury may contribute to burn-related pain. If this is the case, then genetic variations influencing the function of important stress system components, such as the enzyme catechol-O-methyltransferase (COMT), may predict pain severity after thermal burn injury. The authors evaluated the association between COMT genotype and pain intensity in 57 individuals hospitalized after thermal burn injury. Consenting participants at four burn centers were genotyped and completed daily 0 to 10 numeric rating scale pain assessments on 2 consecutive days including evaluation of waking, least, and worst pain. The association between COMT genotype and individual pain outcomes was calculated using a linear mixed model adjusting for sociodemographic and burn injury characteristics. Overall pain (combination of least, worst, and waking pain scores) was significantly higher in patients with a COMT pain vulnerable genotype (6.3 [0.4] vs 5.4 [0.4], P = .037). Individuals with a COMT pain vulnerable genotype also had significantly higher "least pain" scores (3.8 [0.5] vs 2.6 [0.4], P = .017) and significantly higher pain on awakening (6.8 [0.5] vs 5.3 [0.4], P = .004). Differences in worst pain according to genotype group were not significant. COMT pain vulnerable genotype was a stronger predictor of overall pain severity than burn size, burn depth, or time from admission to pain interview assessment. These findings suggest that genetic factors influencing stress system function may have an important influence on pain severity after burn injury. Further studies of genetic predictors of pain after burn injury are needed.

Exploration of CYP450 and drug transporter genotypes and correlations with nevirapine exposure in Malawians.

AIM: Genetic polymorphisms have the potential to influence drug metabolism and vary among ethnic groups. This study evaluated the correlation of genetic polymorphisms with nevirapine pharmacokinetics exposure in Malawians. MATERIALS & METHODS: CYP450 2B6, 2D6, 3A4 and 3A5, ABCB1 and constitutive androstane receptor and pregnane X receptor, were analyzed for polymorphisms in 26 subjects. RESULTS: Allele frequencies (variant) were: CYP2B6 514G>T (0.31) CYP2D6*4 (0.02); CYP2D6*17 (0.35); CYP3A4*1B (0.77); CYP3A5*3 (0.25); ABCB1 2677G>T (0.0), ABCB1 3435C>T (0.21), NR1I3 13711152T>C (0.02), NR1I2 44477T>C (0.10), NR1I2 63396C>T (0.33), NR1I2 6-bp indel (del: 0.17). CYP2B6 516G>T (non-wild-type/wild-type) correlated with nevirapine pharmacokinetic parameters; geometric mean ratios (95% CI): 1.75 (1.27-2.40) for area under the concentration time curve (AUC)(0-12 h), 1.58 (1.03-2.42) for C(0), and 0.53 (0.31-0.91) for clearance. In a multivariable model, nevirapine AUC increased by 1.5% per year of age (p < 0.0001), CYP2B6 516 T allele increased AUC by 92% (p < 0.0001), and CYP3A5*3 decreased AUC by 31% (p = 0.0027). CONCLUSION: Allele frequencies were similar to other sub-Saharan African populations. The T allele for CYP2B6 516 was significantly associated with nevirapine exposure.

Copy number variants in pharmacogenetic genes.

Variation in drug efficacy and toxicity remains an important clinical concern. Presently, single nucleotide polymorphisms (SNPs) only explain a portion of this problem, even in situations where the pharmacological trait is clearly heritable. The Human CNV Project identified copy number variations (CNVs) across approximately 12% of the human genome, and these CNVs were considered causes of diseases. Although the contribution of CNVs to the pathogenesis of many common diseases is questionable, CNVs play a clear role in drug-related genes by altering drug metabolizing and drug response. In this review, we provide a comprehensive evaluation of the clinical relevance of CNVs to drug efficacy, toxicity, and disease prevalence in world populations, and discuss the implication of using CNVs as a diagnostic tool in clinical intervention.

The pharmacological advantage of prolonged dose rate gemcitabine is restricted to patients with variant alleles of cytidine deaminase c.79A>C.

AIM: Controversy exists over the optimal dosing for the nucleoside analogue gemcitabine. A pharmacological advantage is achieved by prolonging infusion times but evidence for a clinical benefit has been conflicting. We hypothesized that polymorphisms in genes involved in gemcitabine accumulation, particularly the cytidine deaminase CDA c.79A>C, may influence the optimal dosing regimen in individual patients. METHODS: DNA was collected from 32 patients participating in a randomized crossover study comparing 30-min with 100-min infusions of gemcitabine. The relationships between seven polymorphisms among three genes (CDA, RRM1 and DCK) and (i) gemcitabine triphosphate accumulation; (ii) gemcitabine-induced toxicity; and (iii) dose delivery were examined for each infusion time and week of administration. RESULTS: There were trends for increased accumulation of gemcitabine-triphosphate (GEM-TP) with the variant alleles of CDA c.79A>C, and RRM1-37C>A and -524T>C but none of these reached statistical significance in a univariate analysis. In a multivariable model there were significant effects of infusion duration and week of administration on GEM-TP accumulation. There were significant interactions between CDA c.79A>C (P=0.01) and RRM1-37C>A (P=0.019) genotypes, infusion time, and arm. More patients with one or two CDA c.79 variant alleles had doses delays (57 vs 13 %, P=0.03) and a pharmacological advantage for prolonged infusion after week 1. CONCLUSION: It is important to consider both pharmacokinetics and pharmacogenetics in optimizing gemcitabine accumulation. This represents a classical interaction between genes and environment and provides support for the consideration of both CDA genotype and infusion duration in development of an individualized dosing strategy.

Irinotecan pharmacogenomics.

Irinotecan is a camptothecin analog used as an anticancer drug. Severe, potentially life-threatening toxicities can occur from irinotecan treatment. Although multiple genes may play a role in irinotecan activity, the majority of evidence to date suggests that variation in expression of UGT1A1 caused by a common promoter polymorphism (UGT1A1*28) is strongly associated with toxicity; however, this link is dose dependent. Variations in other pharmacokinetic genes, particularly the transporter ABCC2, also contribute to irinotecan toxicity. In addition, recent studies have shown that pharmacodynamic genes such as TDP1 and XRCC1 can also play a role in both toxicity and response.

Detection of the G>C SNP and rare mutations in the 28-bp repeat of TYMS using gel-based capillary electrophoresis.

AIMS: Polymorphisms in the 5' regulatory region of the thymidylate synthase gene (TYMS) have been shown to modulate thymidylate synthase expression and are associated with resistance to fluoropyrimidine-based therapies. These polymorphisms include a two repeat (2R) or three repeat (3R) of a 28-bp sequence and a G>C SNP in the second repeat of the 3R allele (TSER*3 G>C). Genotyping methods for the TYMS 5'-UTR polymorphisms have typically involved visualizing PCR and RFLP products on agarose gels. This article describes the use of a robust capillary electrophoresis assay for TYMS 5'-UTR genotyping. MATERIALS & METHODS: As part of pharmacogenetic studies, we performed TYMS genotyping for the 5'-UTR polymorphisms in 314 colorectal cancer patients. A gel-based capillary electrophoresis method, employing a high-resolution gel cartridge on a QIAxcel(®) system, was developed to detect PCR products and RFLP fragment sizes. RESULTS: The high resolution of the capillary electrophoresis technique allowed identification of a 6-bp insertion in the second repeat of the 3R allele in three patients. The frequency of the insertion allele was 0.4% in Caucasians and 1.3% in African-Americans. We also found 3.3% of Caucasian patients were heterozygous for a G>C SNP in the first repeat of the 2R allele, but this allele was not observed in the African-American patients. CONCLUSION: We describe a robust RFLP genotyping technique that employs size discrimination by capillary electrophoresis to genotype the TYMS TSER*3 G>C SNP. The technique also allows identification of a 6-bp insertion in the 3R allele, and we report the allelic frequencies for two uncommon TSER alleles.

Amplification of thymidylate synthetase in metastatic colorectal cancer patients pretreated with 5-fluorouracil-based chemotherapy.

Resistance to 5-fluorouracil (5-FU) represents a major contributor to cancer-related mortality in advanced colorectal cancer patients. Genetic variations and expression alterations in genes involved in 5-FU metabolism and effect have been shown to modulate 5-FU sensitivity in vitro, however these alterations do not fully explain clinical resistance to 5-FU-based chemotherapy. To determine if alterations of DNA copy number in genes involved in 5-FU metabolism-impacted clinical resistance to 5-FU-based chemotherapy, we assessed thymidylate synthetase (TYMS) and thymidine phosphorylase (TYMP) copy number in colorectal liver metastases. DNA copy number of TYMS and TYMP was evaluated using real time quantitative PCR in frozen colorectal liver metastases procured from 62 patients who were pretreated with 5-FU-based chemotherapy prior to surgical resection (5-FU exposed) and from 51 patients who received no pretreatment (unexposed). Gain of TYMS DNA copy number was observed in 18% of the 5-FU exposed metastases, while only 4% of the unexposed metastases exhibited TYMS copy gain (p = 0.036). No significant differences were noted in TYMP copy number alterations between 5-FU-exposed and -unexposed metastases. Median survival time was similar in 5-FU-exposed patients with metastases containing TYMS amplification and those with no amplification. However, TYMS amplification was associated with shorter median survival in patients receiving post-resection chemotherapy (hazard ratio = 2.7, 95% confidence interval = 1.1-6.6; p = 0.027). These results suggest amplification of TYMS amplification as a putative mechanism for clinical resistance to 5-FU-based chemotherapy and may have important ramifications for the post-resection chemotherapy choices for metastatic colorectal cancer.

Institutional profile. UNC Institute for Pharmacogenomics and Individualized Therapy: interdisciplinary research for individual care.

The Institute for Pharmacogenomics and Individualized Therapy (IPIT) at the University of North Carolina at Chapel Hill (NC, USA) is a collaborative, multidisciplinary unit that brings together faculty from different disciplines and crosses the traditional departmental/school structure to perform pharmacogenomics research. IPIT investigators work together towards the goal of developing therapies to enable the delivery of individualized medical care. The NIH-supported Comprehensive Research on Expressed Alleles in Therapeutic Evaluation (CREATE) group leads the field in the evaluation of pathways regulating drug activity, and also provides a foundation for future IPIT research. IPIT members perform bench research, clinical cohort analysis and prospective clinical intervention studies, research on the integration of pharmacogenomic therapy into practice and research to foster global health pharmacogenomics application through the Pharmacogenetics for Every Nation Initiative. IPIT Investigators are actively incorporating a pharmacogenomics curriculum into existing teaching programs at all levels.