Pharmacogenomics, CYP2D6, and Tamoxifen: A Survey of the Reasons Sustaining European Clinical Practice Paradigms.

Tamoxifen is a drug that is often used in the clinical management of breast cancer. CYP2D6 is a key metabolizing enzyme that is involved in the conversion of tamoxifen to its active drug metabolites. CYP2D6 has several alleles that metabolize tamoxifen and other drugs at different rates that can alter therapeutic impact, a characteristic that renders it one of the most studied enzymes in the field of pharmacogenetics. Background and objectives: Portugal has no implemented measures based on pharmacogenomics analysis prior to therapy that might function as a cultural sample control when analyzing the individual and economic factors present in clinical practice paradigms. Therefore, we aim to investigate the impact of CYP2D6 genotyping of the tamoxifen metabolizing enzymes in the clinical management of breast cancer patients. Materials and Methods: Qualitative/quantitative studies regarding the impact of pharmacogenomics in breast cancer; personal interviews in different Portuguese laboratories within hospital setting using a survey. Analysis of data through interviews to management board and/or decision makers from major oncological centers. Results: Reasons for common adoption of pharmacogenomics practice are contradictory and based both in economic factors and cultural/clinical bias. Conclusions: This research study identifies specific cultural and/or clinical bias that act as obstacles to pharmacogenomic implementation and proposes viable courses of action that might bring about change in cultural/medical habits.

Biomarkers for personalized oncology: recent advances and future challenges.

Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells and oncology is a branch of medicine that deals with tumors. The last decade has seen significant advances in the development of biomarkers in oncology that play a critical role in understanding molecular and cellular mechanisms which drive tumor initiation, maintenance and progression. Clinical molecular diagnostics and biomarker discoveries in oncology are advancing rapidly as we begin to understand the complex mechanisms that transform a normal cell into an abnormal one. These discoveries have fueled the development of novel drug targets and new treatment strategies. The standard of care for patients with advanced-stage cancers has shifted away from an empirical treatment strategy based on the clinical-pathological profile to one where a biomarker driven treatment algorithm based on the molecular profile of the tumor is used. Recent advances in multiplex genotyping technologies and high-throughput genomic profiling by next-generation sequencing make possible the rapid and comprehensive analysis of the cancer genome of individual patients even from very little tumor biopsy material. Predictive (diagnostic) biomarkers are helpful in matching targeted therapies with patients and in preventing toxicity of standard (systemic) therapies. Prognostic biomarkers identify somatic germ line mutations, changes in DNA methylation, elevated levels of microRNA (miRNA) and circulating tumor cells (CTC) in blood. Predictive biomarkers using molecular diagnostics are currently in use in clinical practice of personalized oncotherapy for the treatment of five diseases: chronic myeloid leukemia, colon, breast, lung cancer and melanoma and these biomarkers are being used successfully to evaluate benefits that can be achieved through targeted therapy. Examples of these molecularly targeted biomarker therapies are: tyrosine kinase inhibitors in chronic myeloid leukemia and gastrointestinal tumors; anaplastic lymphoma kinase (ALK) inhibitors in lung cancer with EML4-ALk fusion; HER2/neu blockage in HER2/neu-positive breast cancer; and epidermal growth factor receptors (EGFR) inhibition in EGFR-mutated lung cancer. This review presents the current state of our knowledge of biomarkers in five selected cancers: chronic myeloid leukemia, colorectal cancer, breast cancer, non-small cell lung cancer and melanoma.

New developments in hormone receptor-positive disease.

Of more than one million women diagnosed with breast cancer each year, approximately 700,000 have hormone receptor (HR)(+) disease. Although endocrine therapy has revolutionized breast cancer management and substantially improved outcomes in these patients, the optimal management of these patients remains a significant challenge. For instance, the threshold for adding adjuvant chemotherapy is a topic of continuing debate, and the most effective regimens that include endocrine therapy and chemotherapy are still under debate as well. Tumor markers, such as Ki-67, and host markers, such as cytochrome P450 2D6, are being studied as potential tools to offer more tailored adjuvant endocrine therapy. Current research suggests that luminal A and luminal B cancers are two completely different diseases, and work is being performed to better distinguish between these two disease types and deliver more effective therapy to individual patients. This article addresses these important outstanding issues with respect to HR(+) disease.

Cytochrome P450 2D catalyze steroid 21-hydroxylation in the brain.

mRNA of cytochrome P450 21-hydroxylase (P450c21) is expressed in the brain, but little is known about the enzymatic properties of P450c21 in the brain. In the present study, we showed, by using various recombinant cytochrome P450 (CYP)2D enzymes and anti-CYP2D4- or P450c21-specific antibodies, that rat brain microsomal steroid 21-hydroxylation is catalyzed not by P450c21, but by CYP2D isoforms. Rat CYP2D4 and human CYP2D6, which are the predominant CYP2D isoforms in the brain, possess 21-hydroxylation activity for both progesterone and 17alpha-hydroxyprogesterone. In rat brain microsomes, these activities were not inhibited by anti-P450c21 antibodies, but they were effectively inhibited by the CYP2D-specific chemical inhibitor quinidine and by anti-CYP2D4 antibodies. mRNA and protein of CYP2D4 were expressed throughout the brain, especially in cerebellum, striatum, pons, and medulla oblongata, whereas the mRNA and protein levels of P450c21 were extremely low or undetectable. These results support the idea that CYP2D4, not P450c21, works as steroid 21-hydroxylase in the brain. Allopregnanolone, a representative gamma-aminobutyric acid receptor modulator, was also hydroxylated at the C-21 position by recombinant CYP2D4 and CYP2D6. Rat brain microsomal allopregnanolone 21-hydroxylation was inhibited by fluoxetine with an IC(50) value of 2 microm, suggesting the possibility that the brain CYP2D isoforms regulate levels of neurosteroids such as allopregnanolone, and that this regulation is modified by central nervous system-active drugs such as fluoxetine.

Regional and cellular expression of CYP2D6 in human brain: higher levels in alcoholics.

Cytochrome P450 (CYP) 2D6 is expressed in liver, brain and other extrahepatic tissues where it metabolizes a range of centrally acting drugs and toxins. As ethanol can induce CYP2D in rat brain, we hypothesized that CYP2D6 expression is higher in brains of human alcoholics. We examined regional and cellular expression of CYP2D6 mRNA and protein by RT-PCR, Southern blotting, slot blotting, immunoblotting and immunocytochemistry. A significant correlation was found between mean mRNA and CYP2D6 protein levels across 13 brain regions. Higher expression was detected in 13 brain regions of alcoholics (n = 8) compared to nonalcoholics (n = 5) (anovap < 0.0001). In hippocampus this was localized in CA1-3 pyramidal cells and dentate gyrus granular neurons. In cerebellum this was localized in Purkinje cells and their dendrites. Both of these brain regions, and these same cell-types, are known to be susceptible to alcohol damage. For one case, a poor metabolizer (CYP2D6*4/*4), there was no detectable CYP2D6 protein, confirming the specificity of the antibody used. These data suggest that in alcoholics elevated brain CYP2D6 expression may contribute to altered sensitivity to centrally acting drugs and to the mediation of neurotoxic and behavioral effects of alcohol.

Nicotine metabolism and CYP2D6 phenotype in smokers.

We tested the hypothesis that the polymorphic enzyme CYP2D6 is related to nicotine metabolism in 261 healthy subjects enrolling in a smoking cessation clinic. Subjects completed a questionnaire, were given dextromethorphan, and contributed a urine and blood sample. The CYP2D6 phenotype (based on a determination of dextromethorphan and metabolites in an aliquot of overnight urine) and genotype (based on characterization of CYP2D6 variant alleles by a PCR-based method on a subset) were determined. Seventeen poor metabolizers (6.5%) were observed among 261 phenotyped smokers. Nicotine and it chief metabolites, cotinine and trans-3'-hydroxycotinine were measured in the urine and adjusted for pH. All of the nicotine metabolite levels were significantly related to usual and recent smoking. Neither levels of smoking nor nicotine metabolites overall exhibited a relationship to the CYP2D6-deficient metabolizer phenotype. The ratio of nicotine:cotinine + trans-3'-hydroxycotinine, stratified by time since the last cigarette, was unrelated to gender, age, education, race (white/African American), recent alcohol or caffeine consumption, or smoking practices. Subjects in either the lowest quintile or decile metabolic ratio (ultrametabolizers) exhibited a significantly lower nicotine:cotinine + trans-3'-hydroxycotinine ratio after adjustment for recent smoking, pH, and other factors. These data suggest that the polymorphic CYP2D6 gene is not a major contributor to nicotine metabolism in tobacco smokers but may influence the disposition of nicotine in the small subset of the population who are CYP2D6 ultrametabolizers.

Rapid detection of CYP1A1, CYP2D6, and NAT variants by multiplex polymerase chain reaction and allele-specific oligonucleotide assay.

Drugs and carcinogens are excreted from the body after metabolic conversion involving enzymes mediating oxidative metabolism and conjugation. Many of the corresponding genes exhibit functional polymorphisms that contribute to individual cancer susceptibility. To increase the efficiency and to facilitate genotyping, we developed a combined approach (PCR-ASO) which includes multiplex PCR and allele-specific oligonucleotide (ASO) hybridization. PCR primer pairs were used to amplify the following alleles/variants: CYP1A1*1, *2A, *2B; CYP2D6*3, *4; NAT1*4, *3, *10, *11, *14, *15; and NAT2*4, *5A, *5B, *5C, *6A, *7B. The products were dot-blotted and polymorphisms were detected by hybridization with ASO probes for both wild-type and variant sites in parallel. This approach was validated by genotyping DNA samples from a French-Canadian population that was previously analyzed by PCR-RFLP. The variants frequencies were compared with the data on other populations available in the literature. The PCR-ASO assay appears to be simple, efficient, and cost-effective, particularly if a large number of samples are to be screened for several DNA variants. This approach has potential for automation with microplates and robotic workstations for high throughput.

Comparative studies of drug-metabolizing enzymes in dog, monkey, and human small intestines, and in Caco-2 cells.

Drug-metabolizing enzymes were studied in subcellular fractions of dog, monkey, and human small intestines, and in the human adenocarcinoma cell line Caco-2, a commonly used in vitro absorption model. Immunoblot analysis indicated the presence of enzymes related to cytochrome P450 (CYP) 1A1/CYP1A2, CYP2D6, CYP3A, and carboxylesterases (ESs) in human and monkey intestines, and of CYP3A and ES in dog intestines. Catalytically, human and monkey intestines exhibited significant and comparable testosterone 6 beta-hydroxylase, (+)-bufuralol 1'-hydroxylase, and ES activities. In contrast, dog intestine possessed moderate testosterone 6 beta-hydroxylase, much lower ES, and undetectable bufuralol hydroxylase activities. In addition, low tolbutamide methylhydroxylase activity was observed in human and monkey intestines, but not in dog intestines. Of the phase I enzymes investigated, only ES was detected immunologically and functionally in Caco-2 cells. With respect to phase II enzymes, human and monkey intestines contained relatively high intestinal glucuronyltransferase, N-acetyltransferase (NAT), sulfotransferase, and glutathione S-transferase activities. Except for NAT, all phase II enzymes studied were detectable in dog intestines. In Caco-2 cells, acetaminophen sulfation activity was below the limit of detection, whereas all other conjugating activities were evident. Studies of enzyme kinetics and inhibition by known inhibitors of testosterone 6 beta-hydroxylase activity, the major intestinal mono-oxygenase in all species, revealed some similarities between the responsible enzymes. Comparative studies with human liver microsomes suggested the possible involvement of CYP3A enzymes in the intestinal catalysis of testosterone 6 beta-hydroxylation similar to those observed with human hepatic CYP3A. Further studies on ESs, however, revealed multiplicity and species and/or tissue differences in the microsomal and cytosolic enzymes. Based on kinetic studies, monkey intestines and Caco-2 cells possessed NAT activities, with properties similar to those in human intestine and liver. Overall, the results demonstrated that both the preparations of small intestines and Caco-2 cells exhibited significant drug-metabolizing enzyme activities, although several differences were noted between the intestinal enzymes in the animals or in the Caco-2 cells and those found in humans.