Breast cancer: Role of pharmacogenetics in tamoxifen therapy

The role of pharmacogenetics in the personalization of tamoxifen therapy has relevance in the management of breast cancer. Since tamoxifen is a pro-drug, genetic polymorphism in Phase I and II drug metabolizing enzymes involved in the bioconversion of tamoxifen to therapeutically active metabolites is critical in determining therapeutic efficacy and adverse drug reactions of the therapy in breast cancer patients. In this review, the role of pharmacogenetics in the personalization of tamoxifen therapy has been discussed. Since, metabolism of tamoxifen by cytochrome P450 2D6 is significant in determining the therapeutic efficacy of the drug, most of the clinical evidence on tamoxifen pharmacogenetics have been correlated with cytochrome p450 2D6 genetic polymorphism. However, there is discordance in the clinical data, and one of the reasons is the incomplete analysis of all the alleles of cytochrome p450 2D6. International Tamoxifen Pharmacogenomics Consortium has been formed to assess the discordance. There is also clinical evidence associating genetic polymorphism in cytochrome P450 3A, 2C9, 2C19, Uridine diphosphate glucuronosyltransferases (UGT) and sulfotransferases (SULT) with clinical outcome of tamoxifen therapy. However, associations of genetic polymorphism in cytochrome P450 3A, 2C9, 2C19, UGTs and SULT with clinical outcome in populations of different ethnicity are unexplored. Evidence on the association of genetic polymorphisms and the clinical outcome has been summarized. Since cost, statistically significant sample population size, labor, and ethical issues are the major concerns of a pharmacogenetic investigation; the significance of bottom-up approach in pharmacogenetics has been discussed.


Introduction
Cancer is one of the leading causes of death.It has a set back on the psychological and socio-economic status of the patient and family.The incidence and mortality rates of cancer are increasing steadily.It has been estimated that in 2012 8.2 million deaths were caused by cancer. [1]The most common cancers include lung, stomach, prostate, colorectal, liver, esophagus, breast, and cervix.Among them, breast cancer is one of the most common cancers in women and is the leading cause of death after lung cancer.The global estimates of incidence and mortality rates due to breast cancer have increased to 20% and 14%, respectively. [1]It has been observed that the death rate in developed nations is lower than less developed countries.However, the incidence of breast cancer is higher in developed countries than less developed nations and more common in young women than old women. [2]e pathogenesis of breast cancer depends on genetic, environmental, and physiological factors.The formation of DNA adducts is the initial step in the development of cancer.Most of the DNA adducts forms by the activation of the endogenous and exogenous chemicals in the body.The activation and detoxification of the endogenous and exogenous chemicals in the body have been associated with the Phase I and II drug metabolizing enzymes.Most of Phase I and II drug of breast cancer involving mastectomy has been a preferred approach.Besides, chemotherapeutic agents such as the five drug cooper regimen (1960), doxorubicin (1970), anthracyclinebased combinations with cyclophosphamide in metastasis and as adjuvant therapy, combination of cyclophosphamide, adriamycin, and 5-fluorouracil, taxanes for systemic treatment (paclitaxel, docetaxel) anti-estrogen therapies such as tamoxifen, raloxifene, and aromatase inhibitors have shown effective therapeutic efficacy. [4]fore the discovery and development of anti-estrogens such as tamoxifen, surgical procedures such as oophorectomy, hypophysectomy, or adrenalectomy were performed to reduce the secretion of estrogen and progression of cancer.With the discovery and development of tamoxifen, surgical procedures are less preferred in the management of ER positive breast cancer patients.Tamoxifen has reduced the recurrence of breast cancer and improved the overall survival in ER+ early-stage breast cancer patients.To determine the optimal duration of adjuvant tamoxifen clinical trial unit such as adjuvant tamoxifen longer against shorter (ATLAS) have been established under Clinical Trial Service Unit and Epidemiological Studies Unit.The objective of ATLAS is to establish the benefits between the longer (10 years) and shorter (5 years) duration of tamoxifen therapy in patients. [5]e application of hybridoma technology to produce monoclonal antibodies as therapeutic agents has emerged as a significant medical intervention.Monoclonal antibodies such as trastuzumab, pertuzumab (anti HER2-HER3 dimerization antibody), and ado-trastuzumab emtansine (antibody drug conjugate) are the current approaches adopted to treat patients' positive for HER2. [4] the management of triple negative breast cancer, cetuximab, erlotinib, gefitinib, tyrosine-protein kinase kit (c-KIT), phosphatidylinositol 3-kinase -AKT imatinib, sunitinib, dasatinib, anthracyclines, and mitoxantrone are used.Besides, pharmacological inhibitors of the enzyme poly adenosine diphosphate-ribose polymerase (ParP) designed on the concept of synthetic lethality approach are potential therapeutic agents for the treatment of triple-negative breast cancer. [6]wever, there are several challenges in the clinical management of breast cancer.Some of these challenges include heterogeneous nature of the disease, complex pathogenesis (thus poor prognosis of triple-negative breast cancers) and stages of the breast cancer, diagnosis and therapeutic approaches in different sub-types of breast cancer, recurrence, resistance, menopause status of patient (premenopausal, postmenopausal), polypharmacy, demographic characteristics, drug-drug interactions, herb-drug interactions and socioeconomic factors.Thus, poor prognosis of cancer and mortality in patients is irresistible.
With the emergence of pharmacogenomics and pharmacogenetics, the prognosis of breast cancer is easy, and there is a decline in the death rate.The sequencing of human genome and development of robust, sensitive and high-throughput techniques have resolved the problem of interindividual variability and adverse drug reactions of a drug.Thus, in the era of translational science, pharmacogenetics has emerged as the state-of-the-art diagnostics to rationalize the concept of personalized medicine successfully.The concept of personalized medicine is foreseen as a paradigm for a sustainable R and D and business model of pharmaceutical industry.In contrast to the notion of "one drug for all," personalized medicine emphasizes the concept of individualization of the drug therapy.Pharmacogenomics is the key component involved in the individualization of the drug therapy.
Pharmacogenetics of cancer therapy has great relevance.Most of the anticancer drugs are pro-drugs and are biotransformed to an active form mediated by enzymes.The genes encoding for the drug metabolizing enzymes exhibit polymorphism.Hence, the catalytic efficiency of enzymes involved in the catalysis of pro-drugs is variable in individuals.To understand the role of polymorphisms in catalysis of drugs, it is the first important to understand the enzyme activity for each genotype. [7]The existence of functional genetic polymorphism in the population is a major factor for inter-individual variation of a drug therapy.Pharmacogenetic studies on polymorphisms in drug metabolizing enzymes, drug transporters, and drug targets have shown correlations to changes in response and toxicity to commonly prescribed chemotherapeutic treatments of breast cancer.However, most of the pharmacogenetic evidence supports the role of polymorphisms in drug metabolizing enzymes.Genetic polymorphism in drug metabolizing enzymes involved in the metabolism of chemotherapeutic agents of breast cancer such as raloxifene, tamoxifen, 6-mercaptopurine, and irinotecan has been investigated. [8]Among them, tamoxifen pharmacogenetics has relevance in breast cancer treatment.

Pharmacogenetics of Tamoxifen
Tamoxifen is a nonsteroidal anti-estrogen and widely used in the management of premenopausal and postmenopausal metastatic breast cancer.Moreover, in early breast cancer, it is used as an adjuvant therapy and chemopreventive agent for high-risk women.It was approved in 1977 by US-Food and Drug Administration. [9]The trans isomer of tamoxifen is active and has antiestrogenic and estrogenic properties.Clinically, it has been found to be less toxic than other anticancer drugs.Therefore, it is used as the first line drug for postmastectomy adjuvant therapy of early breast cancer and in combination with other drugs for advanced stages.It is also utilized in the treatment of hormone-related ovarian and refractory prostate cancers.
Tamoxifen is a pro-drug and on its intake the pro-drug is principally metabolized by cytochrome P450 2D6, 3A, 2C9 and 2C19 of the liver to therapeutically active metabolites. [10]Evidence on the metabolism of tamoxifen, support the role of cytochrome P450 2D6 as the major enzyme involved in the formation of the therapeutically active metabolites -4-hydroxytamoxifen and endoxifen. [11]However, cytochrome P450 3A, cytochrome P450 2C9, and cytochrome P450 2C19 also contribute in the metabolism of tamoxifen and endoxifen levels in plasma have been associated with clinical outcomes. [12]Both the metabolites 4-hydroxytamoxifen and endoxifen have a higher affinity for the ER than the parent drug.Besides, therapeutically potent metabolites toxic metabolite such as α-tamoxifen, sulfoxy metabolite of tamoxifen (catalyzed by hydoxysteroid sulfotransferases [SULT] 2A1) are also formed.Other Phase I drug metabolizing enzymes such as cytochrome P450 1A1, 1A2, 2B6, flavin-containing monooxygenases (FMO1 and FMO3) also have involvement in the metabolism of tamoxifen.5] Most of Phase I and II drug metabolizing enzymes exhibit genetic polymorphism and it is one of the major reasons for the variation in the clinical outcome of tamoxifen therapy in breast cancer patients. [11]Based on the genotype, breast cancer patients exhibit poor (negligible), intermediate (reduced) and extensive (normal) metabolism of tamoxifen in populations of different ethnicity.Hence, there may be inter-individual variation in therapeutic efficacy and adverse drug reactions such as endometrial cancer and thromboembolic disorders in patients on tamoxifen therapy. [14]The clinical challenge has been investigated by pharmacogenetic studies conducted in populations of different ethnicity.Since cytochrome P450 2D6 is considered to be the principal catalyzing enzyme in tamoxifen metabolism, most of the pharmacogenetic studies explain the significance of cytochrome P450 2D6 polymorphism on tamoxifen therapy in patients with early and advanced stages of breast cancer. [13,16]Breast International Group 1-98 and arimidex, tamoxifen, alone or in combination are randomized clinical trials to assess the role of cytochrome P450 2D6 genotyping in the personalization of tamoxifen therapy. [17]rom genotyping studies in populations of different ethnicity, CYP 2D6 extensive metabolizers are homozygous for *1, *   1]. [11,13,15]he different polymorphisms exhibiting clinically significant outcome such as benefits, disease recurrence, breast cancer specific survival, drug-related hot flashes, and altered in vitro metabolism in comparison to wild type is shown in Table 1 (see supporting material).There is also evidence contrary to the hypothesis and role of cytochrome P450 2D6 genetic polymorphism in the clinical outcome of tamoxifen therapy [Table 1] (see supporting material).It has also been observed that there is discordance in the published observations between cytochrome P450 2D6 genetic polymorphism and the outcome of tamoxifen. [16]International Tamoxifen Pharmacogenomics Consortium has been formed to assess the discordance in the clinical data. [18]The discordance in the data may be a consequence of concomitant use of cytochrome P450 2D6 inhibitors, stereoisomers of endoxifen (active and nonactive forms), inaccurate assessment of endoxifen exposure incomplete analysis of all relevant cytochrome P450 2D6 alleles, disobeying Hardy-Weinberg Equilibrium for allele frequencies and potential contributions of variability in cytochrome P450 3A and cytochrome P450 2C9. [13,16,18]nce, there is no complete association of cytochrome P450 2D6 genetic polymorphism with interindividual variability of tamoxifen metabolism and clinical outcomes. [9,17,10]Besides, Cytochrome P450 2D6, 3A, 2C9 and 2C19 polymorphism of cytochrome P450 1A2, 2B6, FMOs UGTs and SULT may contribute to differences in tamoxifen plasma concentration in individuals. [14,16,19,20]Therefore, the study of the effect of genetic polymorphism of all the enzymes involved in the metabolism of tamoxifen such as cytochrome P450 2C9, 2D6, 2C19, P450 3A, 1A2, 2B6, FMOs, UGTs, and SULT may be useful in the personalization of tamoxifen therapy.
In general, drug therapy involves the interplay of multiple genes.The lack of genetic polymorphism data of different ethnicity and the gap between clinicians and scientists fail to rationalize the concept of personalized medicine.Therefore, a holistic approach overcoming the limitations of the present pharmacogenetic investigations is required.

Bottom-up Approach in Pharmacogenetics of Tamoxifen Therapy
Computational biology has facilitated a deep and a global understanding of the impact of genetic factors on the biology and management of diseases.In drug discovery and development, the "bottom-up approach" involving modeling and simulation are extensively used to study absorption, distribution, metabolism, and excretion of drugs.Hence, safety and efficacy of drugs may be predicted before administration of drugs in human beings.However, in the current pharmacogenetic paradigm, the implementation of "bottom-up" approach is in early stages.
Several clinical investigations have been performed to study the effect of the genetic polymorphism on tamoxifen therapy using      "top-down" approach.However, there are limitations of the "topdown" approach in establishing the correlation of genotype with phenotype and genotype with clinical observations in preliminary investigations.Some of the major limitations are inconsistent and bias data, cost, statistically insignificant sample population size to correlate with possible clinical outcomes, labor-intensive and noncompliance of patients enrolled for the study.Thus, in diseases with complex etiology such as cancer, an effective approach is required for the successful implementation of pharmacogenetics in the personalization of medicines.
In the pharmacogenetics of tamoxifen therapy, "bottom-up" approach may be significant in overcoming discordance in clinical observations.Using the approach interactions of tamoxifen with different alleles of cytochrome P450 2D6 and other relevant enzymes involved in the metabolism of tamoxifen may be evaluated.Furthermore, modeling and simulation may be performed using algorithms such as Simcyp (www.simcyp.com), pharsight to predict the behavior of tamoxifen in virtual populations.Subsequently, clinical trials may be performed to test and validate the novel significant interactions of tamoxifen with enzymes in populations of different ethnicity.
Thus, the early adoption of "bottom-up" approach to the pharmacogenetic study of a drug therapy may be a sustainable approach in the personalization of medicines.

Conclusion
In this review, the current role of pharmacogenetics in tamoxifen therapy has been discussed.The evidence supports the role of cytochrome P450 2D6 genetic polymorphism in tamoxifen therapy.Since, there is discordance in the clinical data involving cytochrome P450 2D6 genetic polymorphism, interaction of tamoxifen with other alleles of cytochrome p450 2D6, and other enzymes has been recommended.To accomplish personalization of tamoxifen therapy bottom up approach has been proposed.

Table 1 : Association of genetic polymorphism with clinical outcome and enzyme activity
Smriti and Manish: Effect of genetic polymorphism of drug metabolizing enzymes on the clinical outcome of tamoxifen International Journal of Molecular & ImmunoOncology ♦ October-December 2016 ♦ Volume 1 ♦ Issue 1 14 (Contd...)