Anne Nguyen

Quantitative effect of CYP2D6 genotype and inhibitors on tamoxifen metabolism: Implication for optimization of breast cancer treatment

N‐Desmethyltamoxifen (NDM), a major primary metabolite of tamoxifen, is hydroxylated by cytochrome P450 (CYP) 2D6 to yield endoxifen. Because of its high antiestrogenic potency, endoxifen may play an important role in the clinical activity of tamoxifen. We conducted a prospective trial in 158 patients with breast cancer who were taking tamoxifen to further understand the effect of CYP2D6 genotype and concomitant medications on endoxifen plasma concentrations.

The Pharmacogenetics Research Network: From SNP Discovery to Clinical Drug Response

The NIH Pharmacogenetics Research Network (PGRN) is a collaborative group of investigators with a wide range of research interests, but all attempting to correlate drug response with genetic variation. Several research groups concentrate on drugs used to treat specific medical disorders (asthma, depression, cardiovascular disease, addiction of nicotine, and cancer), whereas others are focused on specific groups of proteins that interact with drugs (membrane transporters and phase II drug‐metabolizing enzymes). The diverse scientific information is stored and annotated in a publicly accessible knowledge base, the Pharmacogenetics and Pharmacogenomics Knowledge base (PharmGKB). This report highlights selected achievements and scientific approaches as well as hypotheses about future directions of each of the groups within the PGRN. Seven major topics are included: informatics (PharmGKB), cardiovascular, pulmonary, addiction, cancer, transport, and metabolism.

Composite Functional Genetic and Comedication CYP2D6 Activity Score in Predicting Tamoxifen Drug Exposure Among Breast Cancer Patients

Accurate assessment of CYP2D6 phenotypes from genotype is inadequate in patients taking CYP2D6 substrate together with CYP2D6 inhibitors. A novel CYP2D6 scoring system is proposed that incorporates the impact of concomitant medications with the genotype in calculating the CYP2D6 activity score. Training (n = 159) and validation (n = 81) data sets were obtained from a prospective cohort tamoxifen pharmacogenetics registry. Two inhibitor factors were defined: 1 genotype independent and 1 genotype based. Three CYP2D6 gene scoring systems, and their combination with the inhibitor factors, were compared. These 3 scores were based on Zineh, Zanger, and Gaedigks approaches. Endoxifen/NDM‐Tam plasma ratio was used as the phenotype. The overall performance of the 3 gene scoring systems without consideration of CYP2D6‐inhibiting medications in predicting CYP2D6 phenotype was poor in both the training set (r2 = 0.24, 0.22, and 0.18) and the validation set (r2 = 0.30, 0.24, and 0.15). Once the CYP2D6 genotype‐independent inhibitor factor was integrated into the score calculation, the R 2 values in the training and validation data sets were nearly twice as high as the genotype‐only scoring model: (0.44, 0.43, 0.38) and (0.53, 0.50, 0.41), respectively. The integration of the inhibitory effect of concomitant medications with the CYP2D6 genotype into the composite CYP2D6 activity score doubled the ability to predict the CYP2D6 phenotype. However, endoxifen phenotypes still varied substantially, even with incorporation of CYD2D6 genotype and inhibiting factors, suggesting that other, as yet unidentified factors must be involved in tamoxifen activation.

Plasma Letrozole Concentrations in Postmenopausal Women With Breast Cancer Are Associated With CYP2A6 Genetic Variants, Body Mass Index, and Age

The associations between plasma letrozole concentrations and CYP2A6 and CYP3A5 genetic variants were tested in the Exemestane and Letrozole Pharmacogenomics (ELPH) trial. ELPH is a multicenter, open‐label prospective clinical trial in women randomly assigned (n ≈ 250 in each arm) to receive 2 years of treatment with either oral letrozole (2.5 mg/day) or oral exemestane (25 mg/day). CYP2A6 and CYP3A showed effects on letrozole metabolism in vitro. DNA samples were genotyped for variants in the CYP2A6 and CYP3A5 genes. Plasma letrozole concentrations showed high interpatient variability (>10‐fold) and were associated significantly with CYP2A6 genotypes (P < 0.0001), body mass index (BMI) (P < 0.0001), and age (P = 0.0035). However, CYP3A5 genotypes showed no association with plasma letrozole concentrations. These data suggest that CYP2A6 is the principal clearance mechanism for letrozole in vivo. CYP2A6 metabolic status, along with BMI and age, may serve as a biomarker of the efficacy of letrozole treatment or a predictor of adverse effects.

Estrogen Receptor Genotypes Influence Hot Flash Prevalence and Composite Score Before and After Tamoxifen Therapy

PURPOSE Hot flashes are common and frequently lead to drug discontinuation among women prescribed tamoxifen. We determined whether genetic polymorphisms in estrogen receptors (ESRs) alpha and beta (ESR1 and ESR2, respectively) are associated with tamoxifen-induced hot flashes. PATIENTS AND METHODS We determined ESR1 PvuII and XbaI and ESR2-02 genotypes in 297 women who were initiating tamoxifen. One-week hot flash diaries were collected to calculate a hot flash score (frequency x severity) before and 1, 4, 8, and 12 months after starting tamoxifen. RESULTS Approximately 80% of 297 participants reported hot flashes before or during the first year of tamoxifen. After 4 months of tamoxifen, premenopausal women who did not receive adjuvant chemotherapy had a four-fold increase in hot flash score (from 5.9 to 23.6; P = .003) compared with a 1.17-fold increase (from 19.6 to 23; P = .34) in those who received chemotherapy. In premenopausal women, increased number of ESR1 PvuII and XbaI CG alleles was associated with higher baseline hot flash scores compared with those who had other haplotypes (P = .0026). At 4 months, postmenopausal women with ESR1 PvuII CC and ESR2-02 GG genotypes had 4.6 times increases in hot flash scores than other postmenopausal women (56 v 12; P = .0007). Women who had the ESR2-02 AA genotype were significantly less likely to experience tamoxifen-induced hot flashes than women who carried at least one ESR-02 G allele (hazard ratio, 0.26; 95% CI, 0.10 to 0.63; P = .001). CONCLUSION Knowledge of menopausal status, prior chemotherapy, and ESR genotype may help predict which women are most likely to suffer hot flashes during tamoxifen treatment.

Comparison of Subjective and Objective Hot Flash Measures Over Time Among Breast Cancer Survivors Initiating Aromatase Inhibitor Therapy

Objective:Hot flashes are valuable indicators of physiological condition and drug effect; however, subjective and objective measures do not always agree. No study has examined both subjective and objective hot flashes in women prescribed aromatase inhibitors. The study (1) compared subjective and objective hot flash measures, (2) examined changes in subjective and objective hot flashes over time, and (3) evaluated predictors of change in hot flashes in aromatase inhibitor-treated women. Methods:Participants (n = 135) were enrolled in a randomized clinical trial comparing exemestane and letrozole for the treatment of breast cancer. Hot flashes were assessed before the start of the drug therapy and 1, 3, and 6 months later. Participants wore a sternal skin conductance monitor for 24 hours or longer at each time point. With each perceived hot flash, women pressed an event button and rated intensity and bother in a paper diary. Results:Participants had a mean age of 60 years and were mainly white (92%). Across time points, monitor hot flashes were (1) significantly more frequent than diary and/or event button flashes (P < 0.05) and (2) moderately correlated with subjective measures (0.35 < r < 0.56). Monitor hot flashes did not significantly change over time with aromatase inhibitor therapy, whereas both diary and event button frequencies significantly varied but in dissimilar patterns (51% nonlinear). No consistent predictors of hot flashes across measures or time points were identified. Conclusions:Findings indicated dissimilarities between subjective and objective measures of hot flashes. Despite statistical significance, there was little clinically meaningful change in hot flashes after initiating aromatase inhibitor therapy.

Sequence diversity and functional characterization of the 5′-regulatory region of human CYP2C19

Cytochrome p4502C19 (CYP2C19) plays an important role in drug biotransformation and has been shown to be genetically polymorphic. While polymorphisms in the coding region that have large effects on activity are well described, until recently, a lack of knowledge of the promoter sequence has hindered efforts to study it. Genetic variants in the promoter region have not been described and factors that influence its gene expression via promotor regulation remain largely undefined. We have cloned and sequenced 1.8 kb of the human CYP2C19 promoter. This promoter contains a number of putative transcription factor sites, including HepG2-specific factor 1, glucocorticoid response element, estrogen receptor element, constitutive androstane receptor and peroxisome proliferator-activated receptor. Sequencing of DNA obtained from 67 individuals identified eight single nucleotide polymorphisms within this region. No sequence of a known human pregnane X receptor response element was found in this section of the CYP2C19 promoter, despite the known effect of rifampin on the expression of this gene. A plasmid containing the 1.8-kb CYP2C19 promoter coupled to a luciferase reporter gene has been constructed and demonstrated to be functional and sensitive to induction by omeprazole in HuH7 cells. Nested deletions of CYP2C19 promoter were generated and the ability of serial promoter deletion constructs to activate luciferase expression in the HepG2 cell line was analysed. These data make possible future studies to elucidate the molecular mechanisms by which CYP2C19 can be induced in clinical settings and the consequences of genetic variability in its promoter.

A Mixture Model Approach in Gene-Gene and Gene-Environmental Interactions for Binary Phenotypes

In translational research, a genetic association study of a binary outcome has a twofold aim: test whether genetic/environmental variables or their combinations are associated with a clinical phenotype, and determine how those combinations are grouped to predict the phenotype (i.e., which combinations have a similarly distributed phenotype, and which ones have differently distributed phenotypes). The second part of this aim has high clinical appeal, because it can directly facilitate clinical decisions. Although traditional logistic regression can detect gene-gene or gene-environmental interaction effects on binary phenotypes, they cannot decisively determine how genotype combinations are grouped to predict the phenotype. Our proposed mixture model approach is valuable in this context. It concurrently detects main and interaction effects of genetic and environmental variables through a likelihood ratio test (LRT) and conducts phenotype cluster analysis based on genetic and environmental variable combinations. The theoretical distribution of the proposed mixture models likelihood ratio test is robust not only to small sample size but also to unequal sample size in various genotype and environmental subgroups. Hypothesis testing through a likelihood ratio test results in a fast algorithm for p -value calculations. Extensive simulation studies demonstrate that mixture model, overall test in logistic regression, and Monte Carlo based logic regression constantly possess the best power to detect multi-way gene/environmental combinations. The mixture model approach has the highest recovery probability to recover the true partition in the simulation studies. Its applications are exemplified in interim data analyses for two cancer studies.

Genome-wide discovery of genetic variants affecting tamoxifen sensitivity and their clinical and functional validation

BACKGROUND Beyond estrogen receptor (ER), there are no validated predictors for tamoxifen (TAM) efficacy and toxicity. We utilized a genome-wide cell-based model to comprehensively evaluate genetic variants for their contribution to cellular sensitivity to TAM. DESIGN Our discovery model incorporates multidimensional datasets, including genome-wide genotype, gene expression, and endoxifen-induced cellular growth inhibition in the International HapMap lymphoblastoid cell lines (LCLs). Genome-wide findings were further evaluated in NCI60 cancer cell lines. Gene knock-down experiments were performed in four breast cancer cell lines. Genetic variants identified in the cell-based model were examined in 245 Caucasian breast cancer patients who underwent TAM treatment. RESULTS We identified seven novel single-nucleotide polymorphisms (SNPs) associated with endoxifen sensitivity through the expression of 10 genes using the genome-wide integrative analysis. All 10 genes identified in LCLs were associated with TAM sensitivity in NCI60 cancer cell lines, including USP7. USP7 knock-down resulted in increasing resistance to TAM in four breast cancer cell lines tested, which is consistent with the finding in LCLs and in the NCI60 cells. Furthermore, we identified SNPs that were associated with TAM-induced toxicities in breast cancer patients, after adjusting for other clinical factors. CONCLUSION Our work demonstrates the utility of a cell-based model in genome-wide identification of pharmacogenomic markers.BACKGROUND Beyond estrogen receptor (ER), there are no validated predictors for tamoxifen (TAM) efficacy and toxicity. We utilized a genome-wide cell-based model to comprehensively evaluate genetic variants for their contribution to cellular sensitivity to TAM. DESIGN Our discovery model incorporates multidimensional datasets, including genome-wide genotype, gene expression, and endoxifen-induced cellular growth inhibition in the International HapMap lymphoblastoid cell lines (LCLs). Genome-wide findings were further evaluated in NCI60 cancer cell lines. Gene knock-down experiments were performed in four breast cancer cell lines. Genetic variants identified in the cell-based model were examined in 245 Caucasian breast cancer patients who underwent TAM treatment. RESULTS We identified seven novel single-nucleotide polymorphisms (SNPs) associated with endoxifen sensitivity through the expression of 10 genes using the genome-wide integrative analysis. All 10 genes identified in LCLs were associated with TAM sensitivity in NCI60 cancer cell lines, including USP7. USP7 knock-down resulted in increasing resistance to TAM in four breast cancer cell lines tested, which is consistent with the finding in LCLs and in the NCI60 cells. Furthermore, we identified SNPs that were associated with TAM-induced toxicities in breast cancer patients, after adjusting for other clinical factors. CONCLUSION Our work demonstrates the utility of a cell-based model in genome-wide identification of pharmacogenomic markers.

A distinct inflammatory marker pattern in patients with aromatase inhibitor (AI)-induced musculoskeletal symptoms.

Abstract #1129 Background. AI-associated musculoskeletal symptoms (AIMSS) have been reported in up to 50% of treated patients. Inflammatory processes can occur in cancer patients, and inflammatory mediators including interleukins, interferons, MMPs and immunoregulatory molecules have been associated with estrogen signaling. Ceramide and sphingosine-1-phosphate (S1P) are lipid mediators with roles in inflammation. We hypothesized that AIMSS may develop in the context of increased circulating inflammatory markers.
 Methods. Cases (n=24) were selected from 150 early stage ER-positive breast cancer patients who were enrolled on a prospective multicenter randomized trial of exemestane or letrozole. Changes in pain and function during therapy were assessed prospectively, and those patients whose symptoms exceeded a predefined threshold were referred for detailed rheumatologic evaluation. Controls (n=30), who did not develop AIMSS that exceeded the threshold, were matched for age, ethnicity, and prior chemotherapy. Serum samples collected at baseline and 1 and 6 mo following AI initiation were assayed in a blinded manner. The majority of inflammatory molecules were quantified in serum using an addressable laser bead multiplex assay (BMD, Paris) on a Luminex S100 analyzer (Austin, TX). Ceramide and S1P were alkaline chloroform/methanol-extracted from serum, 32P-labeled, and quantified using autoradiography. Statistical comparisons between cases and controls for each factor were performed using the non-parametric Mann-Whitney test or by log transforming the data and using unpaired two-tailed t tests.
 Results. The mean serum concentrations for the majority of assayed factors (IFNγ, IL1Rα, IL2/4/5/7/10/15/17, IL2R, IL12p40, IP10, MIP1α, MIG, bFGF, HGF, GCSF, and GMCSF) were significantly lower at baseline in cases compared to controls (p Conclusions. Subjects who develop AIMSS appear to have an underlying anergic cytokine phenotype at baseline. During AI therapy, patients with AIMSS develop higher levels of S1P, a potential inflammatory mediator known to induce COX2 and NFκB. These hypothesis-generating results suggest a potential mechanism underlying the development of AIMSS, distinct from other rheumatologic conditions. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1129.