Cancer Treatment Reviews

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1 Cancer Treatment Reviews 38 (2012) Contents lists available at SciVerse ScienceDirect Cancer Treatment Reviews journal homepage: Pharmacogenetics of anti-estrogen treatment of breast cancer Marzia Del Re a, Angela Michelucci b, Paolo Simi b, Romano Danesi a, a Division of Pharmacology, Department of Internal Medicine, University of Pisa, Italy b Cytogenetics and Molecular Genetics Unit, University Hospital, Pisa, Italy article info abstract Article history: Received 1 April 2011 Received in revised form 8 August 2011 Accepted 12 August 2011 Keywords: Tamoxifen Aromatase inhibitor CYP2D6 CYP19A1 Estrogen receptor Polymorphisms Pharmacogenetics A major effort is underway to select genetic polymorphisms potentially relevant to the clinical efficacy and safety of endocrine treatment of breast cancer. Genetic factors of the host that affect the metabolism of tamoxifen, a widely used drug for the adjuvant treatment of breast cancer, have received particular attention. Cytochrome P450 isoform 2D6 (CYP2D6) is a key step in the metabolism of tamoxifen to its active moiety endoxifen. Women with functionally deficient genetic variants of CYP2D6 who are given drugs that inhibit CYP2D6 are exposed to low endoxifen plasma levels and may enjoy reduced benefits from tamoxifen treatment. Therefore, CYP2D6 status may be an important predictor of the benefits of tamoxifen to an individual; unfortunately, the data are not uniformly concordant, and definitive evidence that would suggest the routine analysis of CYP2D6 before commencing tamoxifen treatment is not yet available. Recent research has focused on the role UDP-glucuronosyltransferases, a family of metabolizing enzymes that play an important role in the metabolic clearance of tamoxifen and of the aromatase inhibitors as well, and how interindividual differences in these enzymes may play a role in the clinical outcome upon administration of anti-estrogen treatment. In conclusion, whether a pharmacogenetic profile should be obtained prior to initiating tamoxifen therapy is currently a matter of debate, although summing up all the scientific evidence available on this issue it appears that the genetic screening would be an useful support for clinical decision making in selected patients. Ó 2011 Elsevier Ltd. All rights reserved. Introduction Breast cancer is the most common malignancy of women in the Western world and it is associated with an enormous amount of morbidity and mortality worldwide. According to estimates by the World Health Organization International Agency for Research on Cancer, approximately 1.29 million women were diagnosed and over 400,000 women died with breast cancer in Clinical relevance of ER, PR and HER2/neu Corresponding author. Address: Division of Pharmacology, Department of Internal Medicine, University of Pisa, 55, Via Roma, Pisa, Italy. Tel.: ; fax: address: r.danesi@med.unipi.it (R. Danesi). The first major classification of breast cancer is the identification of the estrogen (ER) and progesterone (PR) receptor status. More than two thirds of breast cancers are positive on immunohistochemical staining to the estrogen receptor (ER), so hormonal therapies are the backbone of treatment for the large majority of patients with breast cancer. Both estrogen synthesis and activity through the ER have been targeted by therapies to control endocrine-dependent breast cancer; however, not all women whose tumors express ER derive benefit from hormonal therapies. Differentiating breast cancers, just on the basis of ER and PR status vastly improves treatment efficacy 26 ; the ER-positive (ER+) tumors are dependent on estrogen signaling for their growth and replication and they can be treated by antiestrogen therapy with either tamoxifen or an aromatase inhibitor (AIs). The ER-negative (ER ) tumors, instead, are not estrogen-dependent and will not respond to antiestrogen therapy but are more responsive to first line chemotherapeutic combinations such as paclitaxel, 5-FU, doxorubicin and cyclophosphamide (T/FAC). 62 Expression of the PR is highly correlated with ER expression but the effect of PR status on treatment seems to be less important than that of ER. 18 Tamoxifen is the gold standard treatment for early and advanced disease and its activity on ER+ cancers is considered the first form of molecular targeted therapy. Recently, the introduction of aromatase inhibitors either in metastatic or in adjuvant setting has added a novel and more active tool in the breast cancer treatment. 66 Another well characterized tumor marker is the human epidermal growth factor receptor 2 (HER2/neu). HER2 has been identified as a marker of poor prognosis and increased cancer aggressiveness 59 and treatment of HER2+ tumors has changed significantly with the use of trastuzumab, a monoclonal antibody specific for the HER2/neu protein /$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi: /j.ctrv

2 M. Del Re et al. / Cancer Treatment Reviews 38 (2012) Predictive markers of treatment efficacy Despite these important clinical advances, however, not all patients respond to endocrine therapy and a substantial number of patients who respond will develop subsequent disease progression or recurrence while on therapy. Pharmacogenetics may account in part for the interpatient variability in treatment response to endocrine therapies than that is commonly observed. Variations in genes that encode for enzymes associated with drug metabolism or elimination, or a drug target, can lead to individual differences in drug distribution, metabolism, activity, and toxicity. 22,86 SNPs are often found in the promoter region or in the coding region of the gene and they can cause, respectively, an altered expression of the enzyme or amino acid substitution in the translated protein (non-synonymous SNP), resulting in a variant protein that have increased or decreased activity relative to the wild-type protein. It has also been demonstrated that a SNP in a coding region that does not change the amino acid sequence (synonymous SNP) can impact protein folding kinetics and create an improperly folded and functionally variant protein. 47 The identification of SNPs that considerably alter the function or expression of proteins involved in the pharmacokinetics or pharmacodynamics of drugs and their ultimate effect on efficacy and safety is the aim of pharmacogenetics, with the purpose of an individualized and personalized therapy. Treatment for breast cancer is constantly evolving as new technologies, therapies, and strategies are discovered and the individualization of breast cancer treatment promises to increase effectiveness and decrease drug-related toxicities while reducing health care costs. The role of estrogens in hormone-dependent breast cancer Aromatase (CYP19A1) When ovarian estrogen synthesis ceases at the menopause, estrogens continue to be synthesized in different tissue compartments from circulating androgens. The key pathway is aromatization of androstenedione into estrone, probably accounting for as much as 90% of the total estrogens synthesized in a postmenopausal woman (Fig. 1). Aromatase, also called estrogen synthetase, is a cytochrome P450-dependent enzyme symbolized as CYP19A1 and catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogens. The aromatase enzyme may also use testosterone as a substrate to convert testosterone into estradiol (Fig. 1); however, since the circulating testosterone levels are lower than those of androstenedione, and aromatase has lower activity on testosterone than androstenedione, the contribution of this pathway to estrogen synthesis is only modest. Upon receiving electrons from NADPH-cytochrome P450 reductase, aromatase converts androstenedione and testosterone to estrone and estradiol, respectively (Fig. 1). The adrenals are the main source of circulating androgens in postmenopausal women, while the potential role of the postmenopausal ovary remains controversial. Although only one gene encodes for the aromatase enzyme in both reproductive and non-reproductive tissues, this gene contains at least 10 different promoters. A possibility of tissue-specific aromatase inhibition exists, because these promoters are expressed differently in normal tissues and in breast cancer, and the promoters are stimulated by different ligands. Aromatase is present in many tissues including female breast, skin, muscle, fat and nerve, where it may contribute to sex-specific differences in cellular metabolism. Estrogens are involved in the development and growth of breast tumors; approximately 60% of premenopausal and 75% of postmenopausal breast cancer patients have estrogen-dependent carcinomas. 4 The breast is influenced by estrogen from peripheral sources as well as through aromatase activity in its stroma and epithelial cells. High levels of estradiol may be a risk factor for breast cancer. 90 There is also evidence of elevated levels of aromatase activity and mrna expression in stromal cells in breast cancer, as well as increasing evidence of cross-talk between estrogens and androgens in regulating gene expression in the breast. 71 Moreover, circulating estrogens can compete with androgens for binding to sex hormone-binding globulin, and it is generally assumed that sex hormone-binding globulin synthesis is regulated by and is a reflection of the estrogen/androgen balance. 27 ER a and b b-estradiol (E2) exerts its activity by binding to the two ERs normally present in the mammary gland, ERa and ERb. In particoular ERa is present in about 40% to 70% of breast cancers and it is considered a predictive factor of endocrine therapy responsiveness; the role of ERb, however, is less clear. The binding of E2 on ERa induces a conformational modification and allows the link of co-activators; in contrast, the binding of tamoxifen to ERa increases the association to co-repressor proteins which actively inhibit gene transcription. The activity of tamoxifen and of other selective ER modulators (SERMs) depends on the balance between co-activators and co-repressor proteins, differently represented in tissue and in breast cancer. 11,74 Endocrine treatment of breast cancer: Tamoxifen Tamoxifen CYP2D6 Endoxifen Androstenedione CYP19A1 17-β-HSD Testosterone 17-β-HSD Estrone Estradiol Transcription Proliferation CYP19A1 ER Anastrozole Letrozole Exemestane Raloxifene Toremifene Tamoxifen is the most commonly used agent for the treatment of ER+ breast cancer and belongs to the class of selective estrogen receptor modulators (SERMs). The drug was approved by the US Food and Drug Administration in 1977 for the treatment of metastatic breast cancer and subsequently approved for use in the adjuvant setting; in addition, the Early Breast Cancer Trialists Collaborative Group demonstrated that five years of therapy with tamoxifen reduced the annual risk of breast cancer recurrence by 39%. 21 Substantial interindividual variation exists in steady state levels of tamoxifen and its metabolites following standard dosing. 43 A considerable portion of patients with metastatic disease and patients receiving adjuvant tamoxifen finally relapse, suggesting that the benefit from the therapy is not uniform. Cancer cell Pharmacogenetics of tamoxifen metabolism Fig. 1. Schematic representation of action of steroid hormones and their inhibitors on breast cancer cells. 17-b-HSD: 17b-hydroxysteroid dehydrogenase; ER: estrogen receptor. Tamoxifen is metabolized via the cytochrome P450-mediated pathway to metabolites that show variable potencies toward the

3 444 M. Del Re et al. / Cancer Treatment Reviews 38 (2012) Tamoxifen 4-OH-tamoxifen 2D6, 2B6, 2C9, 2C19, 3A4 2D6, 3A4, 3A5, 1A2, 2C9, 2C19 FMOs Tamoxifen N-oxide 3A4, 3A5 N-Desmethyl tamoxifen SULT1A1 Sulphate Endoxifen 2D6 O NH 2 Sulphate SULT1A1 3A4, 3A5 HO Norendoxifen Fig. 2. Schematic representation of metabolic pathways of tamoxifen. : uridine 5 0 -diphospho-glucuronosyltransferases; SULT1A1: sulfotransferase 1A1; FMOs: flavincontaining monooxygenases. CYP isoforms are reported in the figure by omitting the cytochrome p450 abbreviation. ER (Fig. 2). N-desmethyltamoxifen, resulting from CYP3A4/5-mediated metabolism, is the major primary metabolite, accounting for 90% of tamoxifen oxidation; whereas 4-OH-tamoxifen, mediated by CYP2D6 activity, is a minor metabolite (Fig. 2). Both N-desmethyl-tamoxifen (via CYP2D6) and 4-OH-tamoxifen (via CYP3A4/5) are secondarily metabolized to 4-OH-N-desmethyl-tamoxifen (endoxifen); 4-OH-tamoxifen and endoxifen are important active metabolites, exhibiting similar potency. 75 Norendoxifen is a potent inhibitor of CYP19A1 and is produced by CYP3A4/5. 54 Tamoxifen metabolites are inactivated by sulfotransferases 1A1 (SULT1A1), or by the UDP-glucuronosyltransferases (UGT), including UGT1A8, 1A10, 2B7, 2B15, and 2B17 isoforms (Fig. 2). In addition, CYP1B1, 2B6, and 2C19 may be responsible for the isomerization of trans- 4-OH-tamoxifen to its weakly estrogenic cis-isomer form, a reaction that could be associated with drug-resistant phenotypes. 67 CYP3A4 Because of many of the tamoxifen-metabolizing enzymes are polymorphic, genetic variations may account for interindividual or interethnic differences in tamoxifen-related outcomes. Genetic variants responsible for tamoxifen metabolism have been studied: A CYP3A4 promoter variant, CYP3A4 1B ( 392A>G) has been associated with cancer phenotype, 50,81 but no studies to date have linked CYP3A4 1B with altered tamoxifen metabolism, although it has been reported to confer a three-fold increased risk of endometrial cancer in tamoxifen treated women. 13 CYP3A5 A CYP3A5 genetic polymorphism, CYP3A5 3 (6986A>G), results in markedly decreased CYP3A5 activity, although several studies have failed to show its association with tamoxifen metabolism or breast cancer outcomes. 46,80 However, in a study on postmenopausal patients treated with adjuvant tamoxifen, who were homozygous for the CYP3A5 3C variant, displayed significantly improved recurrence-free survival. 85 These conflicting findings underscore the complexity of tamoxifen metabolism and resistance mechanisms and suggest that the evaluation of variants representing different interacting mechanisms may be more informative than evaluating single variants. 39 CYP2C19 The hypothesis that CYP2C19 681G>A and 636G>A variants, known for their lack of enzyme activity, are associated with an increased breast cancer mortality rate was studied in 80 patients given tamoxifen. CYP2C19 681AA carriers were associated with a significantly longer breast cancer survival rate than patients with the wild-type (hazard ratio 0.26), suggesting that CYP2C19 genotype may possibly be a predictive factor for survival in breast cancer patients using tamoxifen. 70 CYP2D6 More than 80 allelic variants have been described in CYP2D6. A selection of them is presented in Table 1. A commonly accepted classification system designates individuals homozygous for two wild-type CYP2D6 alleles as CYP2D6 1/ 1, and refers to such subjects as extensive metabolizers (EMs). Individuals with one or two variant alleles that encode enzymes with reduced or null activity are designated intermediate metabolizers (IMs) and poor metabolizers (PMs), respectively; a minority of population may carry multiple copies of CYP2D6 alleles and consequently are called ultrarapid metabolizers (UMs), such individuals have increased enzymatic activity compared to CYP2D6 1/ 1. The inci-

4 M. Del Re et al. / Cancer Treatment Reviews 38 (2012) Table 1 CYP2D6 gene variants. Variant Functional effect Reference 3 Non-functional (homozygotes or heterozygotes had decreased plasma endoxifen concentrations on adjuvant tamoxifen) Jin et al Non-functional (allele carriers had more recurrences and significantly worse disease-free survival) Goetz et al Gene deletion and lack of enzyme activity (allele carriers had significantly worse event-free survival with adjuvant tamoxifen) Schroth et al Non-functional (homozygotes or heterozygotes had decreased plasma endoxifen concentrations after adjuvant tamoxifen) Jin et al Reduced activity (allele carriers had worse recurrence outcomes with adjuvant tamoxifen and metastatic breast cancer patients had shorter TTP with tamoxifen) Lim et al. 51 ;Xu et al Reduced activity (allele carriers had increased recurrences, decreased relapse-free survival and significantly worse event-free survival with adjuvant tamoxifen) Schroth et al. 72 dence of SNPs in CYP2D6 varies by race and ethnicity. 7 African- Americans, for example, have more than twice the median frequency of nonfunctional alleles compared with Africans (14.5% versus 6.3%). 29 However, evidence from published studies evaluating CYP2D6 variants as predictive factors of tamoxifen efficacy and clinical outcome are conflicting. In a study on 618 breast cancer patients treated with adjuvant tamoxifen, CYP2D6 4, 5, 10 and 41 alleles were genotyped and the recurrence free survival (RFS) was calculated based on inferred CYP2D6 phenotypes using Kaplan Meier and Cox proportional hazard models, adjusted for nodal status and tumor size. Patients with at least one reduced function CYP2D6 allele (60%) or no functional alleles (6%) had a non-significant trend for worse RFS (hazard ratio (HR) 1.52). For postmenopausal women on tamoxifen monotherapy, the HR for recurrence in patients with reduced functional alleles was However, RFS analysis limited to four common CYP2D6 allelic variants was not significant (P = 0.39). 78 Another study investigated CYP2D6 1, 4, 5, 6b/c, 9, 10, 41 and UM functional variants and the correlation with breast cancer specific survival (BCSS) in 6640 DNA samples from patients with invasive breast cancer, of wich 3155 cases had received tamoxifen therapy. There were 312 deaths from breast cancer, in the tamoxifen-treated patients, with over years of cumulative follow-up. The association between genotype and BCSS was evaluated using Cox proportional hazards regression analysis. In tamoxifen treated patients, there was weak evidence that the poor-metaboliser variant, CYP2D6 6, was associated with decreased BCSS (P = 0.02; HR = 1.95). No other variants, including CYP2D6 4, previously reported to be associated with poorer clinical outcomes, were associated with differences in BCSS, in either the tamoxifen or non-tamoxifen groups. 1 In another study the impact of CYP2D6 4 genotype on progression-free survival (PFS) and time to progression (TTP) was evaluated in 493 patients with ER+ tumors and tamoxifen therapy longer than 6 months. No significant difference in TTP or PFS between the CYP2D6 4 and 1 genotype groups was found. In a subgroup of patients treated with chemotherapy the CYP2D6 4 poor metabolizers had a tendency towards a shorter mean TTP; in this group the mean TTP and the PFS were 1 year in the CYP2D6 4/ 4 group, 6.3 years in the 1/ 4 group and 4.97 years in the 1/ 1 group. 76 A study on 206 patients receiving adjuvant tamoxifen and on 280 control untreated subjects (71 months median follow-up) genotyped for several polymorphisms of CYP2D6, CYP2C19, CYP2B6, CYP2C9, and CYP3A5, tamoxifen-treated patients carrying the deficient CYP2D6 alleles 4, 5, 10, 41 had significantly more recurrences of breast cancer, shorter relapse-free periods, and worse event-free survival rates compared with carriers of functional alleles. Patients with the high enzyme activity promoter variant CYP2C19 17 had a more favorable clinical outcome than carriers of 1, 2, and 3 alleles. 72 In a study on 190 postmenopausal breast cancer patients treated with adjuvant tamoxifen, CYP2D6 4 homozygotes had significantly worse RFS and DFS and lower incidence of hot flashes compared with women with either one or no CYP2D6 4 variant alleles. 35 This was also confirmed in a cohort of 486 patients treated with tamoxifen who were carriers of the CYP2D6 4, CYP2D6 5, CYP2D6 10, and CYP2D6 41 alleles and had significantly worse survival, whereas no association was found among the untreated. 72 Concomitant administration of CYP2D6 inhibitors, in addition to genetic variants, may affect tamoxifen-related outcomes, converting an extensive metabolizer to a phenotypic poor metabolizer. CYP2D6 inhibitors include antidepressants such as fluoxetine and paroxetine, whereas moderate/weak inhibitors include cimetidine, amiodarone, and haloperidol. 33 CYP2D6 extensive metabolizers who took potent CYP2D6 inhibitors had lower plasma endoxifen concentrations and poorer RFS than those who did not. 5,34 Studies correlating CYP2D6 variants with tamoxifen outcome were unable to assess the predictive importance of endoxifen pharmacokinetics, probably because CYP2D6 influences the exposure to several active metabolites that collectively account for tamoxifen pharmacodynamics. The levels of tamoxifen, endoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were measured in 1370 samples from patients with ER+ disease. Breast cancer outcomes were not associated with the concentration levels of tamoxifen, 4-OH-tamoxifen, and N-desmethyltamoxifen. 56 The usefulness of CYP2D6 testing before tamoxifen use is still a matter of debate and no specific recommendation has been issued with respect to the testing. It is not clear whether women who are intermediate or poor CYP2D6 metabolizers should avoid tamoxifen or receive an higher dose and it is not established a treatment algorithm based on CYP2D6 genotype. Several studies have evaluated pharmacogenetic influences on tamoxifen metabolism, efficacy, and safety; however, the development of third-generation aromatase inhibitors, developed in the early 1990s, including two non-steroidal triazole derivatives (anastrozole and letrozole) and one steroidal derivative (exemestane), has provided an alternative strategy for managing hormone-responsive breast cancer in postmenopausal women. 8 However, a recent study on 119 patients treated with tamoxifen 20 mg/day for at least 4 months and not on any strong CYP2D6 inhibiting medications demonstrated that plasma levels of endoxifen in CYP2D6 poor metabolizer were 8 10 times lower than extensive metabolizers (EM) and that increasing the tamoxifen dose to 40 mg/daily significantly increased endoxifen levels in intermediate (IM) and poor metabolizers (PM). 45 Assuming that the concentration of the active metabolite at the target tissue is of potential crucial importance for an optimal outcome of treatment, a reasonable strategy is to adapt the drug dose based on CYP2D6 genotype, especially when a patient bearing an IM or PM phenotype is treated with strong or moderate CYP2D6 inhibitors, including buproprion, duloxetine, paroxetine, fluoxetine, quinidine, amiodarone, terbinafine, haloperidol, indinavir or ritonavir. SULT1A1 and UGT The enzymes responsible for elimination and inactivation of tamoxifen and its metabolites through conjugation with either a sulfate (SULT) or a glucuronide (UGT) moiety may also have functionally relevant genetic variants. The SULT1A1 2 variant (638G>A, Arg213His) encodes for a protein with reduced activity. However, two studies exploring its association with tamoxifen pharmacoki-

5 446 M. Del Re et al. / Cancer Treatment Reviews 38 (2012) netics were negative, 46,85 whereas a third study on 160 patients reported conflicting results on a greater risk of death in tamoxifen-treated patients. 63 In vitro studies suggested that the UGT1A4 variant Leu48Val shows increased glucuronidation activity against tamoxifen and its metabolites, although the clinical significance is still unexplored. 77 A nonsynonymous polymorphism in UGT2B15 (UGT2B15 2; 253G>T) in a putative substrate binding domain has been assessed in adjuvant tamoxifen-treated breast cancer patients and found that subjects possessing SULT1A1 2/ 2 and either UGT2B15 1/ 2 or UGT2B15 2/ 2 had a significantly reduced 5-year survival. 64 Pharmacogenetics of ER Most patients with tumor progression, despite tamoxifen treatment, continue to express ER in malignant tissue suggesting that mechanisms other than loss of ER expression, including polymorphisms of ER, are responsible for treatment failure. Differential expression of the exon 5 deletion splice variant of the ER (del5- ER) has been proposed to account for this acquired resistance, but has not been substantiated in large studies. 24 Numerous variants have been identified in the ER gene in DNA from breast tumors. No functional consequence has been ascribed to the majority of these variants. However, two rare point mutations in the ER, Asp351Tyr and Tyr537Asn have been shown to be associated with altered response to estradiol and antiestrogens. 84 ERa and ERß genes are highly polymorphic, and some polymorphisms have been related to breast cancer risk, 65 or to gain in bone mineral density (BMD) in patients treated with SERM. Moreover, the relative contribution of the two receptors to breast cancer development and hormonal therapy responsiveness is not well understood. An association between (CA) n polymorphism of ERa and BMD in 21 breast cancer patients treated with tamoxifen was demonstrated. CA tandem repeats are located in intron 5 of ERß gene, and their functional significance is unknown, although they may affect mrna levels. Allele variants range from 14 to 24 repeats. Breast cancer patients who are (CA) 21 allele-carriers (about 27% among Asians) show a significantly higher increase of BMD than non-carriers, at 12 months after the start of tamoxifen therapy. 89 Moreover, a number of repeats inferior to 23 have been related to high risks of breast cancer development in a study of 153 invasive breast tumors and 318 controls. 2 Taken together, these evidences suggest a possible role of CA repeats in tamoxifen responsiveness of breast cancer patients. Two common variants are PvuII (rs ) and XbaI (rs ) polymorphisms, which are 45 bp apart and located approximately 400 bp upsteam of exon 2 of the ER-a gene. A study on 1069 patients showed that the PvuII and the (GT) n polymorphisms of ERa gene are strong prognostic indicators of survival in women with ER + breast cancers. 6 In particular, PvuII polymorphism is located on intron 1 and two major variants are known. Among ER + breast cancer patients, P-allele carriers have a significantly lower probability of death than non-carriers. The PvuII polymorphism shows a prevalence of 36.4% in heterozygous (Pp) and 19.8% in homozygous (pp) carriers among Caucasians. 65 Recently, evidences have been provided for a functional effect of the PvuII polymorphism. 38 They found that the P-allele produces a binding site for myb transcription factor. Thus, P-allele carriers may have higher transcription activity of ER, and may be potentially associated with particular benefit from SERMs therapy. (GT) n is a highly polymorphic microsatellite sequence located on 5 0 -end of ERa gene. Its functional significance is unknown, but it may be related to transcriptional regulation of ERa gene. ER+ breast cancer patients carrying one or two (GT) 23 repeats show significantly lower probability of death. On the contrary, (GT) 18 carriers show a worse prognosis. (GT) 23 and (GT) 18 prevalence is respectively 15.3% and 19.5%. 6 Finally, PvuII and XbaI polymorphism, both located in intron 1 at -397 bp and -351 bp from exon 2 have been related to response to SERMs, including raloxifene, measured in terms of bone mineral density gain. 37 No functional role for XbaI polymorphism is known; XbaI prevalence is 36% in heterozygous and 29.3% in homozygous carriers among Caucasians. 65 Rather than ER status per se, it appears that methylation of the ERa gene is a more stringent predictor of clinical response in patients treated with tamoxifen. Epigenetic events like DNA methylation and hystone deacetilation are disrupted during breast cancer progression, determining abnormal expression patterns in cancer cells. In particular, ERa promoter methylation can silence this gene, rendering breast cancer cells resistant to endocrine therapy. Moreover, treatment of ER breast cancer cells with demethylating agents may revert this acquired resistance. 32 Recent evidences suggest that ERa methylation status, as well as abnormal methylation of other genes, may be predictive indicators of tamoxifen responsiveness and breast cancer recurrence. 25,87 In particular, 148 breast tumor samples were studied using MethyLight PCR, which allows an exact quantification of methylation status in specific DNA regions. They found that patients treated with tamoxifen with high levels of ERa methylation showed better survival than tamoxifen-treated patients with low levels of ERa methylation. In this study, methylation levels were determined on an intronic sequence, although we still do not know if the biological significance of intronic sequence methylation is the same as promoter methylation. Moreover, MethyLight PCR is able to find abnormally methylated cancer DNA in tumor samples as well as in blood samples. 25 Since DNA methylation changes from blood samples during therapy were not monitored, it is not clear whether tamoxifen response is associated or not with epigenetic modifications of ERa gene. Endocrine treatment of breast cancer: Aromatase inhibitors In post-menopausal women, the major source of estrogen is the peripheral synthesis of estrone and estradiol by aromatase, which is the target of specific inhibitors. Aromatase inhibitors including anastrozole, exemestane and letrozole, exert their activity by inhibiting estrogens production. They proved to be active in advanced and early breast cancers in postmenopausal hormone receptor positive patients 30,31 and have demonstrated improved activity compared to tamoxifen in various clinical settings. 41,44 Randomized trials in metastatic setting have shown improved efficacy of third generation aromatase inhibitors compared to megestrol acetate in breast cancer patients after failure of tamoxifen. In first line trials, letrozole, anastrozole and exemestane have demonstrated an improved TTP compared with tamoxifen. 10 Arthralgia has emerged as a major symptom in breast cancer survivors on aromatase inhibitors for adjuvant hormonal therapy. 12,14 About an half of patients on aromatase inhibitors attribute arthralgia to this class of medication. 16,57 Aromatase inhibitor-associated arthralgia (AIAA) results in decreased function, premature discontinuation and sub-optimal adherence to therapy. 17,61 This symptom has the potential to impair quality of life and drug effectiveness. Although the pathophysiology of AIAA remains unclear, estrogen suppression is hypothesized to play an important role, since aromatase inhibitors block the final step in estradiol and estrone synthesis. 23 Natural menopause has been associated with increased joint aches and stiffness; symptoms are most prominent during the late menopausal transition when marked falls in circulating estrogen levels occur. 19 Among breast cancer survivors, clinical risk factors associated with AIAA include shorter time since menopause and chemotherapy exposure, which further diminishes residual ovarian estrogen production. 16,57 Thus, estrogen suppression, the main effect of aromatase inhibitor exposure, appears linked to arthralgia.

6 M. Del Re et al. / Cancer Treatment Reviews 38 (2012) Pharmacogenetics of aromatase inhibitors and treatment efficacy Table 2 CYP19A1 gene variants. Variant Functional effect Reference 115T>C/27142C>T Decreased enzyme activity Ma et al. 55 (Arg 39 Cys 264 ) 27142C>T (Cys 264 ) Decreased enzyme activity Ma et al C>T (Thr 364 ) Decreased enzyme activity Ma et al UTR (161A>C) Increased complete response rate and time to progression in postmenopausal metastatic breast cancer patients treated with letrozole Colomer et al UTR (19C>T) Increased enzyme activity (enhanced risk of breast cancer) Kristensen et al. 48 CYP19A1 is a complex gene with many polymorphic and splicing variants and some polymorphisms have been related to breast cancer risk 48 or to an abnormal activity of aromatase. 55 Pharmacogenetic associations pertaining to their efficacy and toxicities have not been well described, although efforts are ongoing to elucidate these mechanisms. In many studies selected CYP19A1 polymorphisms (Table 2) have been investigated for their possible association with the therapeutic efficacy of aromatase inhibitors and with sex hormone levels or risk for estrogen-dependent diseases. 3,20,36,48,52,60,73,79,83 Although the results of these studies are controversial, they suggest an important role for genetic variation in aromatase function and, as a result, in estrogen production. One of the most frequently studied CYP19A1 polymorphism is the (TTTA) n tandem repeat in intron 4. Some authors reported that the (TTTA) 12 allele (prevalence of 4% among breast cancer patients, 1% in general population) is associated with significantly higher risk of breast cancer. 49 Moreover, the (TTTA) 12 polymorphism has been related to higher aromatase activity and plasma estrogen levels in postmenopausal women. 68 The non-synonymous Arg264Cys polymorphism is associated with reduced (about 40%) protein levels compared to wild type and this polymorphism shows a prevalence of 2.5% among Caucasians. Functional characterization in a study on 240 DNA samples showed that the Cys264, Thr364, and double variant Arg39Cys264 allozymes show decreased activity compared with wild-type; the Arg39Cys264 allozyme also displayed a significant increase in inhibitor constant for letrozole, suggesting a relative resistance of this protein variant. 55 Alternative splicing of CYP19A1 mrna produces at least 8 tissue specific transcripts. Abnormal splice variant production has been related to aromatase overexpression in breast cancer. 9 The effects of a C/ T SNP in the 3 0 UTR of CYP19 (rs10046) were studied on tumor mrna levels and splice variants and correlated with clinical parameters and risk of breast cancer in a population of 481 patients and 236 controls. This polymorphism resulted associated with increased risks of breast cancer and the frequency of TT genotypes was significantly higher in patients versus controls (P = 0.007) particularly among those with stage III and IV disease (P = 0.004) and with tumors larger than 5 cm (P = 0.001). A significant association between the variant T allele and the level of aromatase mrna in tumors was observed (P = 0.018), as well as with a switch from adipose promoter to ovary promoter (P = 0.004). It is conceivable that the T allele may increase the stability of ovary-specific CYP19A1 mrna, thus producing a high aromatase activity breast cancer. 48 In a study on 95 postmenopausal women with stage II III ER/PgR+ breast cancer treated with neoadjuvant letrozole, patients were evaluated for correlation between response to treatment and three polymorphisms of CYP19A1, one in exon 7 (790C>T, rs700519) and two in the UTR region (19T>C, rs10046 and 161C>A, rs4646), on DNA obtained from peripheral blood. Thirty-five women (36.8%) achieved a radiological response to letrozole. The histopathological and immunohistochemical parameters, including hormonal receptor status, were not associated with the response to letrozole and only the genetic variant AC/AA of the rs4646 polymorphism was associated with poor response to letrozole (P = 0.03). Eighteen patients (18.9%) reported a progression of the disease and those patients carrying the genetic variants AC/AA of rs4646 presented a shorter PFS than the patients homozygous for the reference variant (P = ). This effect was especially significant in the group of elderly patients not operated after letrozole induction (P = 0.009), suggesting that the rs4646 polymorphism identifies a subgroup of stage II-III ER/PgR [+] patients with poor response to neoadjuvant letrozole and poor prognosis. 28 In a study on breast cancer subjects, genomic DNA obtained from 52 women pre- and postaromatase inhibitor treatment and genomic DNA obtained from 82 samples of breast cancer and 19 samples of normal breast tissue was subjected to resequencing. No differences in CYP19A1 sequence were observed between tumor and germ-line DNA in the same patient and a total of 48 SNPs were identified including four novel SNPs. For genotype-phenotype association studies, the levels of aromatase activity, estrone, estradiol, and tumor size in patients pre- and post-aromatase inhibitor treatment were determined. Two linked SNPs (rs and rs in the 5 0 -flanking region of CYP19 exon 1) were significantly associated with a greater change in aromatase activity after aromatase inhibitor treatment. 82 In a follow-up study in 200 women with early-stage breast cancer who were treated with adjuvant anastrozole, these same two SNPs were also associated with higher plasma estradiol levels in patients pre- and post-aromatase inhibitor treatment. 82 More recently, a small study in 67 patients described the association of the CYP19A UTR variant rs4646 with higher complete response rate and improved TTP in postmenopausal, hormone receptor positive, metastatic breast cancers treated with letrozole. 15 Another study on 94 patients evaluated 13 CYP19A1 polymorphisms in women receiving neoadjuvant letrozole and derived a predictive model comprising eight most informative polymorphisms, including rs4646, which could predict letrozole response at 4 months with 86% accuracy. 53 Some data also report the association of CYP19A1 Val 80 and [TTTA] n polymorphisms and the risk of breast cancer in BRCA carriers and noncarriers. The study consisted of 958 cancer cases and 931 healthy controls, including 474 carriers and 1415 noncarriers. Val 80 G/G genotype was associated with significantly increased risk of breast cancer compared with the Val 80 A/A genotype in BRCA1 carriers aged <50 years (P = 0.032) but not in BRCA2 carriers or noncarriers of any age. A similar association, although non-statistically significant, was found between Val 80 polymorphism and estrogen receptor-negative status of the breast tumors. A common haplotype composed of the Val 80 G allele and three haplotype-tagging SNPs (rs727479, rs10046, and rs4646) in the CYP19 coding region showed a trend to association with breast cancer risk in BRCA1 carriers aged <50 years. In addiction, published data show higher estrogen levels with higher repeats in [TTTA] n found in linkage disequilibrium with Val 80 allele. 69 Pharmacogenetics of aromatase inhibitors and treatment tolerability A cross-sectional study was performed in 390 postmenopausal women with stage 0 III breast cancer receiving adjuvant aromatase inhibitor therapy. Patient-reported AIAA was the primary outcome. DNA was genotyped for candidate CYP19A1 polymorphisms and serum estrogen levels were evaluated by radioimmunoassay. Multivariate analyses were performed to examine associations between AIAA and genetic variants controlling for possible confounders. A percentage of 50.8% of patients reported AIAA. Women carrying at least one eight-repeat allele had lower odds of AIAA

7 448 M. Del Re et al. / Cancer Treatment Reviews 38 (2012) (P = 0.008) after adjusting for demographic and clinical covariates. Estradiol and estrone were detectable in 47% and 86% of subjects on aromatase inhibitors, respectively. Although these post-aromatase inhibitor levels were associated with multiple genotypes, they were not associated with AIAA. In multivariate analyses, women with more recent transition into menopause (less than 5 years) were significantly more likely to report AIAA than those more than ten years in post-menopause (P < 0.001). 58 Another study tried to identify single nucleotide polymorphisms associated with musculoskeletal adverse events in women treated with aromatase inhibitors for early breast cancer. Selected patients were those displaying grade 3 or 4 musculoskeletal adverse events or those who discontinued treatment for any grade of musculoskeletal adverse events within the first 2 years. The study included 293 cases and 585 controls. A total of 551,358 SNPs were analyzed, followed by imputation and fine mapping of a region of interest on chromosome 14. Four SNPs on chromosome 14 had the lowest P values (2.23E-06 to 6.67E-07). T-cell leukemia 1A (TCL1A) was the gene closest ( bp) to the four SNPs. Functional genomic studies revealed that one of these SNPs (rs ) created an estrogen response element and that TCL1A expression was estrogen dependent, was associated with the variant SNP genotypes in estradioltreated lymphoblastoid cells transfected with ERa gene and was directly related to interleukin 17 receptor A (IL17RA) expression. 42 pain. A postmenopausal patient with osteoporosis may be treated with an aromatase inhibitor and a bisphosphonate if she is a CYP2D6 poor metaboliser, but may be better served with tamoxifen if she is an extensive metaboliser. Concerning the aromatase inhibitors, the scientific evidence on the usefulness of genetic screening is much less consistent and awaits further confirmation; therefore no recommendations on their clinical use can be made at this time. Finally, it is possible and desirable that in the future a thorough genetic analysis of the tumor and the host will help clinicians to identify the most appropriate drug and dosage to be administered to the patient. Future pharmacogenetic studies will need to focus on CYP19A1 and aromatase inhibitor and, with the availability of genome-wide screening platforms at reasonable cost, also on integration of multiple drug pathways to allow a more comprehensive analysis of genetic factors influencing drug efficacy and toxicity. Conflict of interest statement The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript that could inappropriately influence the work presented herein. Conclusions and perspectives Hormonal treatment of receptor positive breast cancer is of paramount importance for the survival of patients with breast cancer. However, despite the large number of data that, at least indirectly, provide evidence of the potential clinical importance of many factors of variability that affects the metabolism and target interaction of anti-estrogen drugs, little use is made in the clinic of the available testing to assess such variability. Due to the lack of convincing data arising from randomized controlled trials and the lack of a cost-effectiveness analysis of applying genetic screening to breast cancer patients, the clinician perception of the added value of screening patients for known biomarkers is poor. However, it should be pointed out that the scientific relevance of combining together the large amount of both preclinical and clinical data, although retrospectively collected, is substantial. Indeed, it appears that some breast cancer patients taking tamoxifen may not be getting the full benefit of their treatment because of their metabolizer status and/or they have also been taking selective serotonin reuptake inhibitors or other drugs that inhibit transformation of tamoxifen into its active metabolites. Therefore, the clinician should consider the importance of discussing with the patient the potential benefit of genetic screening of known CYP2D6 allelic variants in order to choose the most appropriate dose of tamoxifen, particularly if she is taking a known inhibitor of CYP2D6 because the administration of such drugs in a CYP2D6 poor metabolizer may reduce the production of endoxifen below the level of clinical activity. Also, genetic screening may challenge the superiority of aromatase inhibitors over tamoxifen demonstrated in clinical trials. Indeed, the stratification of patients based on their CYP2D6 genotype and phenotype may reveal a similar clinical efficacy between the two drug classes if poor metabolizers are removed from the analysis. Another issue that can be addressed by genetic screening is the occurrence of adverse drug reactions (ADR). Tamoxifen can cause hot flashes and a vaginal discharge; a patient that cannot tolerate such ADR may be screened for CYP2D6 genotype and the drug dose may be reduced if she is found CYP2D6 extensive metabolizer. On the contrary, aromatase inhibitors increase the risk of osteoporosis and fractures and are also associated with sometimes severe joint References 1. Abraham JE, Maranian MJ, Driver KE, et al. CYP2D6 gene variants: association with breast cancer specific survival in a cohort of breast cancer patients from the United Kingdom treated with adjuvant tamoxifen. Breast Cancer Res 2010;12:R Anghel A, Raica M, Marian C, et al. 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