The patient experience

The impact of improved treatments for the management of hormone-sensitive breast cancer extends beyond clinical responses. Thanks to appropriate literature and access to the internet, patient awareness of treatment options has grown and patients are now, in many cases, able to engage their oncologists in informed conversations regarding treatment and what to expect in terms of efficacy and safety. Indeed, patients realize that although there is no cure for metastatic disease, treatment can greatly reduce the risk of progression and in the adjuvant setting, where treatment is administered with a curative intent, current treatment options reduce the risk of relapse. The approval of letrozole throughout the breast cancer continuum has provided patients with many reassuring options. The improvement in outcome with letrozole is achieved without a detrimental effect on overall quality of life. Adverse events such as hot flushes, arthralgia, vaginal dryness, and potential osteoporosis are most significant from the patient’s perspective, and it is important that caregivers pay attention to patients experiencing these events, as they can impact compliance unless effectively explained and managed. The major benefits of letrozole are to improve prospects for long-term survivorship in the adjuvant setting and to delay progression and the need for chemotherapy in the metastatic setting.
Introduction
The diagnosis of breast cancer is a devastating blow for women and is associated with depression, anxiety, and a range of other psychological problems, such as self-blame and negative perception of body-image [1–5]. Newly diagnosed metastatic breast cancer (MBC) is also associated with high levels of psychological morbidity, particularly for younger women [6]. Prevention of relapse is a long-term therapeutic imperative, but the impact of therapy on quality of life (QOL) also needs to be taken into consideration when planning treatment strategies.
All therapeutic modalities for early breast cancer (mastectomy or lumpectomy, radiation, chemotherapy, antibody therapy and endocrine therapy) can have a significant impact on patients’ QOL both in the short-term and in the transition period from primary treatment to long-term survivorship [7–9]. Receiving good quality information about prognosis, treatment options, side effects, and risks of breast cancer recurrence is, therefore, important for patients diagnosed with breast cancer [10–13].
The third-generation aromatase inhibitor (AI) letrozole was introduced a decade ago, but at that time, a limited amount of information on breast cancer and its treatment was available to patients. Since then, the international oncology community has made major advances in its knowledge about breast cancer biology, the individualization of treatment options, and communication with patients. Greater understanding of genetics and breast cancer risk has improved approaches for counseling individuals about their susceptibility [14]. In women who develop breast cancer, the individualization of treatment is becoming more sophisticated through the use of biomarkers as prognostic and predictive factors. These include human epidermal growth factor receptor (HER) 2 [15, 16], urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 [17, 18], mRNA expression patterns [19, 20], and, more recently, genetic profiling techniques [21, 22]. Individualizing treatment is also made easier by the ability to more accurately determine the patient’s risk factors and prognosis; for example, by using the Adjuvant! Online program [23–25]. Recognizing breast cancer overtreatment has also contributed to tailoring treatment according to individual risk [26–30]. Oncology teams have facilitated patient involvement in selecting the most appropriate therapy [24, 31, 32] and have improved the provision of patient information, support, and counseling [33, 34].
At the same time, patients began to organize effectively and expand their access to knowledge of new drugs and treatment paradigms [35–37]. Patients have learned to be informed so that they can have constructive dialogues with their doctors and feel they understand and contribute to treatment decisions [38–42]. Patients who take an active role in the decision-making process perceive that they have a treatment choice, in contrast to those who prefer a shared or passive role [43]. They know they cannot be told at any time during the course of their disease that they are or are not cured; they recognize that breast cancer is a chronic illness. Patients are now empowered with hope and the knowledge that breast cancer can be managed. Moreover, increased survival rates have prompted greater interest in the QOL of breast cancer survivors [44–46].
Better education and improved access to screening has resulted in fewer patients being diagnosed with advanced breast cancer at their first presentation, and consequently, mortality has decreased [29, 47–49]. In addition, more effective adjuvant therapy with third-generation AIs has significantly decreased the risk of recurrence as compared with the previous standard of care based on tamoxifen [50–53]. However, there remains a long-term risk of breast cancer recurrence over time [54, 55]. Hormone receptor-positive (HR+) breast cancer is particularly challenging because of the persistent risk of recurrence with this chronic “smoldering” disease. The Early Breast Cancer Trialists Collaborative Group meta-analysis showed that in untreated women with the same nodal status, the breast cancer death rate is greater for patients with estrogen receptor-negative (ER−) versus ER+ tumors in the first 5–6 years, but substantially lower for ER− versus ER+ tumors over the next 10 years [56]. Similarly, Saphner reported that beyond 5 years, the risk of recurrence was higher for patients with HR+ tumors than for those with HR− tumors (P = 0.00002) [54].
Patients may experience stress after medical therapy is ended because they feel they have lost a safety net [57, 58]. This fear is understandable and justifiable in view of the persistent risk of recurrence for HR+ breast cancer. An analysis of the MA.17 trial of extended adjuvant therapy recently showed an increasing risk of disease recurrence over time in patients treated with placebo after discontinuing tamoxifen [59]. Not surprisingly, therefore, patients may be willing to receive long-term therapy (extended adjuvant) to prevent recurrence provided that the benefits outweigh the risks and QOL is maintained [60]. Patients will try to tolerate treatment-related adverse events if there is the prospect of achieving a “cure,” i.e. remaining free of relapse during their lifetime.
Tamoxifen was the mainstay of breast cancer therapy, but its time-dependent efficacy and serious adverse events created a need for new therapies [61–63]. The suppression of estrogen was shown to be greater and more selective with third-generation AIs than with first- and second-generation compounds [64], and this has resulted in better clinical outcomes and improved tolerability [65]. This review examines the clinical use of the third-generation AI letrozole from the patient’s perspective and assesses how it has improved treatment outcomes across the breast cancer continuum, including advanced or MBC, extended and initial adjuvant therapy, and neoadjuvant therapy.
Metastatic setting
Endocrine therapy is the first-choice treatment for women with HR+ breast cancer without acute life-threatening symptoms and should be administered for as long as possible before switching to cytotoxic regimens. Endocrine therapy is preferred to cytotoxic chemotherapy because of its more favorable safety profile [66]. Thus, extending the time to the initiation of chemotherapy is important with endocrine therapy, because a considerable proportion of patients who progress to chemotherapy will experience toxic side effects without gaining benefit [67]. The most common acute side effects of chemotherapy, such as leukopenia, alopecia, and nausea and vomiting, are significantly increased in women receiving combination regimens compared with single agents [68]. Chemotherapy can also adversely affect certain aspects of QOL, notably increasing fatigue [69], and some regimens may be associated with severe or life-threatening complications such as cardiac failure [70]. A meta-analysis to review the evidence and determine whether chemotherapy or endocrine therapy has the most beneficial effect on treatment outcomes (survival, response rate, toxicity, and QOL) concluded that in women with HR+ MBC, a policy of treating first with endocrine therapy rather than chemotherapy is recommended except in the presence of rapidly progressive disease [71].
Prior to the introduction of third-generation AIs, postmenopausal patients with hormone-responsive MBC had few endocrine therapy options other than tamoxifen [62]. Second-line endocrine agents were limited by safety concerns, including weight gain or the risk of thromboembolism with megestrol acetate [72, 73] and cardiovascular toxicity with aminoglutethimide [65]. Randomized controlled trials demonstrated that letrozole is better tolerated and more effective than these second-line endocrine agents [65, 72, 73]. Furthermore, letrozole has also demonstrated superior early survival compared with tamoxifen as first-line therapy in postmenopausal patients with MBC [74]. Importantly, longer time to disease progression with letrozole versus tamoxifen was achieved without increased time with adverse events and resulted in more quality-adjusted survival for patients on letrozole [75]. Maintaining functional ability is an important goal for patients treated in this setting. A subanalysis by different sites of metastatic lesion and Karnofsky Performance Scores also showed the superiority of letrozole compared with tamoxifen in patients with nonvisceral metastases, with visceral metastases without liver involvement, and with liver metastases [76]. From the patient’s perspective, it is important to receive the most effective therapy first-line; therefore, letrozole represents a more attractive option than tamoxifen for postmenopausal women.
Pharmacologic and clinical differences exist between third-generation inhibitors and should be considered in the selection of the most appropriate endocrine therapy [64, 77]. Data from a small randomized crossover trial in 72 postmenopausal women with HR+ MBC showed that overall QOL was significantly better with letrozole than with anastrozole (P = 0.002 for mean total Functional Assessment of Cancer Treatment-endocrine symptoms) [78]. Furthermore, letrozole was significantly better tolerated overall than anastrozole. Less nausea, fewer hot flashes, and less abdominal discomfort resulted in almost twice as many patients preferring letrozole to anastrozole [78].
While letrozole is an appropriate first-line therapy for the majority of patients with hormone-responsive MBC, a small subset of patients with HR+ HER2+ tumors have high-risk disease and are candidates for early treatment with chemotherapy plus the anti-HER2 monoclonal antibody trastuzumab [79]. A meta-analysis of 12 studies involving 2,379 patients with MBC demonstrated that HER2+ tumors are less responsive than HER2− tumors to endocrine treatment (overall relative risk 1.42; 95% confidence interval, 1.32–1.52; P < 0.00001) [80]. Evidence from preclinical models suggests that trastuzumab may overcome relative resistance to endocrine therapy [81], providing the rationale for combining anti-HER2 and endocrine therapies in the clinical setting. Recently, the first published results of an AI in combination with an anti-HER2 antibody (i.e. trastuzumab) show that the combination demonstrated durable responses for at least 1 year in 25% of patients [82]. Preliminary results from a phase 3 trial (TrAstuzumab in Dual HER2 ER-positive Metastatic breast cancer) demonstrated that the combination of trastuzumab with an AI (anastrozole) was more effective than anastrozole alone in postmenopausal patients with HR+ HER2+ MBC [83]. Another phase 3 trial, which is enrolling more than 1,200 patients in this setting and has just finished recruitment, is investigating letrozole in combination with lapatinib, a dual inhibitor of HER2 and HER1 tyrosine kinases [84]. The strategy of combining letrozole with trastuzumab (as investigated in the evaluation of Letrozole combined with Trastuzumab trial), or another HER2-directed therapy, may allow patients with HR+, HER2+ tumors to safely delay the initiation of cytotoxic chemotherapy.
Adjuvant therapy
Patients receiving adjuvant therapy expect their treatment to prevent breast cancer recurrence and offer the prospect of cure. However, even modest gains in survival are sufficient to make adjuvant endocrine treatment worthwhile for premenopausal women with early-stage breast cancer [85]. This benefit threshold also appears to apply to women with a higher risk of recurrence for whom adjuvant chemotherapy is indicated [86]. Adjuvant therapy should be tailored to suit the needs of individual patients based on their clinical risk factors, attitudes, and personal life circumstances [25]. However, it is important to recognize that individual preferences cannot always be fully explained on the basis of treatment-related determinants and patient or clinical characteristics [87].
The Anastrozole versus Letrozole: Investigation into Quality of Life study compared the effects of anastrozole and letrozole on estrogen levels, QOL, lipids, and bone health [88]. A total of 185 postmenopausal women with invasive breast cancer were randomized to receive adjuvant therapy with either 12 weeks of letrozole followed by 12 weeks of anastrozole or vice versa. Of the patients who have completed the 12 weeks of treatment (n = 146), 50 (34%) had a preference for neither drug, 50 (34%) preferred anastrozole, and 46 (32%) preferred letrozole. Both estradiol (E2) and estrone sulfate levels (E1S) were significantly lower on letrozole than on anastrozole (P < 0.000001). Thus, 2.5 mg of letrozole reduces circulating E2 and E1S levels to a significantly greater degree than 1 mg of anastrozole, with no significant difference in patient preference. The greater suppression of estradiol levels might translate into improved clinical efficacy, although further studies, such as the ongoing Femara Anastrozole Clinical Evaluation trial, are required to confirm these findings.
Patients with HR+ tumors need to consider the prospect of life-long adjuvant therapy in view of the persistent risk of disease recurrence [54, 56, 89]. Following the introduction of third-generation AIs, there is much greater choice in endocrine therapy. The challenge for physicians is to select the most appropriate strategy to suit individual patient circumstances. Letrozole first entered the adjuvant setting as a treatment option for patients completing 5 years of tamoxifen [90] and has demonstrated clinical superiority over tamoxifen as initial adjuvant therapy [51] and neoadjuvant therapy [91].
Neoadjuvant therapy
Neoadjuvant endocrine therapy with letrozole is an attractive option for some postmenopausal patients with HR+ early breast cancer [92], including elderly patients who are unable or unwilling to undergo chemotherapy or surgery, and patients with locally advanced HR+ tumors who wish to have breast-conserving surgery but are not suited for preoperative chemotherapy [91, 93]. In a randomized controlled trial, letrozole demonstrated a superior overall objective response rate and rate of breast-conserving surgery compared with tamoxifen as neoadjuvant therapy [91]. Currently, letrozole is the only AI approved in the neoadjuvant setting (in the United Kingdom and 16 other countries worldwide). It provides a reasonable therapeutic alternative to preoperative chemotherapy in postmenopausal women with HR+ disease in clinical situations where the low toxicity of the regimen is considered an advantage, e.g. in women older than 70 years [91]. However, patients need to be aware that a longer course of therapy may be required to achieve an objective response than is the case with neoadjuvant chemotherapy [94]. In one study, neoadjuvant letrozole was safely given over 12 months to postmenopausal women with large operable or locally advanced HR+ breast cancers [94]. A longer treatment course may suit some patients and give them more time to consider their options for surgery, radiotherapy, and chemotherapy.
Neoadjuvant therapy with letrozole could be considered a sensitivity test of endocrine therapy that might be incorporated into strategies to individualize adjuvant treatment according to response [95]. This would provide reassurance to patients that they will be receiving an adjuvant endocrine therapy that has shown activity against their tumor. Objective assessment of the antitumor activity of neoadjuvant letrozole can be made on the basis of response rate and by analysis of predictive biomarkers. In one study, neoadjuvant letrozole was found to inhibit tumor proliferation (determined by the biomarker Ki67) more effectively than tamoxifen [95]. Letrozole was effective independently of HER2 expression status, although the greatest difference between letrozole and tamoxifen was seen in tumors that were HR+ and HER1/2+. Recent evidence has suggested that HER2+ tumors can continue to proliferate despite neoadjuvant letrozole or tamoxifen treatment, which could imply therapeutic resistance that may manifest later in the clinical course of the disease [96].
Biomarker studies may also reveal differences between AIs. In a randomized comparative trial, letrozole and anastrozole significantly reduced proliferation in HR+, HER2+ or HER2− tumors [97]. Decreased proliferation was seen at all Allred ER expression levels with both agents, but only letrozole showed a significant effect in the lower ER cases. In addition, more cases showed a reduction in progesterone receptor (PgR) expression following letrozole than anastrozole [97]. Another study showed evidence of a decrease in HER2 expression after neoadjuvant treatment with letrozole [98].
Although complex, biomarkers may become increasingly important from a patient’s perspective, because they can help to improve the individualization of treatment. Furthermore, biomarkers may be useful to predict the risk of resistance to endocrine therapy and the need to consider alternative approaches in the future, such as combination therapies or cytotoxic chemotherapy. Knowing what to expect next is important for patients. However, further work is necessary to validate the use of clinical markers and biomarkers in the neoadjuvant setting as surrogate end points for long-term outcomes [96].
Early adjuvant therapy
Tamoxifen was the gold standard endocrine therapy for all women with HR+ breast cancer until recent results from large randomized trials challenged this paradigm for postmenopausal women [50, 51, 99]. Oncologists and patients now face an important choice when selecting adjuvant endocrine treatment [66, 100], whether to start with the most potent endocrine therapy (an AI) upfront or to start with tamoxifen and switch to an AI inhibitor after 2–5 years. The ongoing Breast International Group (BIG) 1-98 randomized trial is expected to provide more information on the benefits of switching to letrozole after 2–3 years on tamoxifen and will clarify which is the optimal strategy. The final results are expected to be released in 2008 [51], but until then, the most appropriate endocrine therapy will need to be selected on the basis of currently available evidence. The MA.17 trial has already demonstrated that letrozole treatment is beneficial after 5 years of adjuvant tamoxifen [90, 101, 102].
Patients will ideally wish to receive treatment that provides the greatest long-term efficacy with the lowest risk of adverse events. BIG 1-98 showed that letrozole was significantly more effective than tamoxifen as initial adjuvant endocrine therapy [51]. Moreover, tamoxifen is perceived to be more toxic than AIs, largely because of well-publicized “scares” about increased risk of uterine cancer and thrombosis [61, 103]. These side effects and vaginal bleeding were reported for tamoxifen in BIG 1-98, whereas letrozole was associated with more skeletal events. The overall rate of cardiovascular events was not significantly different between the groups. Letrozole was also shown to be extremely well-tolerated in comparison with placebo in the MA.17 trial [90, 101]. With the exception of adverse events related to suppression of estrogen, there was no difference in adverse events (Table 1).
Table 1Safety profile of letrozole in comparison with placebo (A) and tamoxifen (B) reported in postmenopausal women with early breast cancerPlacebo Acute toxicities reportedTotal number (%), any grade101Letrozole (n = 2,572)Placebo (n = 2,577)Edema571 (22)542 (21)Hypertension130 (5)129 (5)Hot flushes1,486 (58)a1383 (54)Fatigue999 (39)998 (39)Sweating782 (30)760 (29)Anorexia142 (6)a110 (4)Constipation363 (14)382 (15)Diarrhea168 (7)176 (7)Nausea308 (12)314 (12)Vaginal bleeding145 (6)196 (8)aInfection without neutropenia124 (5)112 (4)Arthritis167 (6)137 (5)Hypercholesterolemia418 (16)411 (16)Dizziness458 (18)441 (17)Insomnia166 (6)135 (5)Depression143 (6)131 (5)Headache706 (27)685 (27)Arthralgia651 (25)a532 (21)Myalgia380 (15)a310 (12)Bone pain141 (5)149 (6)Dyspnea161 (6)163 (6)Alopecia126 (5)a89 (3)Vaginal dryness147 (6)129 (5)TamoxifenWorst grade adverse events recorded within first 28 daysTotal number (%), any grade51Letrozole (n = 3,975)Tamoxifen (n = 3,988)CVA or TIA39 (1.0)41 (1.0)Thromboembolic event61 (1.5)140 (3.5)aCardiac event (IHD, CF)162 (4.1)153 (3.8)Other CV event19 (0.5)a8 (0.2)Vaginal bleeding132 (3.3)263 (6.6)Hot flushes1332 (33.5)1516 (38.0)aNight sweats554 (13.9)647 (16.2)aFracture225 (5.7)a159 (4.0)Arthralgia806 (20.3)a491 (12.3)Myalgia254 (6.4)243 (6.1)CV cardiovascular, CVA cerebrovascular accident, TIA transient ischemic attack, IHD ischemic heart disease, CF cardiac failureaSignificant difference
Based on the results from the BIG 1-98 and the Anastrozole and Tamoxifen Alone or in Combination (ATAC) trial, which compared upfront AIs with tamoxifen, it is clear that all postmenopausal women with HR+ breast cancer should be given the opportunity to receive adjuvant use of an AI, and this recommendation is now reflected in internationally recognized treatment guidelines [66, 100, 104, 105]. Patients with HR+ breast cancer considered eligible for adjuvant chemotherapy because of their increased risk for relapse should also be candidates for the most effective adjuvant endocrine strategy [100]. Of note, the BIG 1-98 trial showed that adjuvant letrozole provides significant disease-free survival (DFS) benefits for patients at increased risk of recurrence, specifically patients with node-positive tumors, large primary tumors (>2 cm), and recipients of chemotherapy and also demonstrated a significant reduction in the risk of distant recurrence, a well-known predictor of breast cancer death [51]. Additional analyses of data from BIG 1-98 to determine the predictive value of centrally tested ER, PgR, and HER2 status on the response to letrozole and tamoxifen indicate that there is no difference in subgroups by ER/PR or HER2 status regarding the superiority of upfront letrozole versus tamoxifen [106]. Low-risk patients with HR+ breast cancer who do not receive adjuvant chemotherapy are also candidates for AIs [104] because of the persistent risk of relapse [54, 56].
While it is known that tamoxifen loses effectiveness after 5 years [62, 63], the optimal duration of initial therapy with an AI remains to be determined [107]. The MA.17 randomized controlled trial of extended adjuvant endocrine therapy has already shown that letrozole treatment is beneficial after 5 years of tamoxifen in postmenopausal patients [90, 101], but there are no equivalent data on the use of tamoxifen after initial AI therapy in the adjuvant setting [100]. Furthermore, the efficacy of switching to another AI in patients who discontinue treatment with letrozole is not known. Therefore, patients starting upfront therapy with letrozole will need reassurance that according to present knowledge, treatment can be continued for up to 5 years, and other options will be available down the line. In addition, patients will require information about the potential long-term impact of AIs on bone and the cardiovascular system [107].
According to the current treatment guidelines [66, 100, 104, 105], patients already taking a course of adjuvant tamoxifen may wish to switch to anastrozole or exemestane, as these AIs have demonstrated efficacy in this setting [52, 108, 109]. Although recent data have shown that this sequential adjuvant strategy is associated with a survival advantage [53, 110, 111], it is important to note that these data were obtained from a selected population comprising patients at randomization who were disease-free after 2–3 years of tamoxifen. It is therefore not valid to make a direct comparison with an unselected population treated in trials of upfront AI therapy. The decision to switch endocrine therapy after 2–3 years, i.e. for a sequential tamoxifen–AI strategy, should be based on an individual’s risk for recurrence, risk for osteoporosis, and ability to tolerate tamoxifen. Importantly, BIG 1-98 is expected to provide more information on the benefits of switching to letrozole after 2–3 years of tamoxifen, and will clarify whether upfront or sequential AI use is the optimal strategy. The final results will be released in 2008 [51].
Extended adjuvant (including late extended adjuvant) therapy
Many patients fear breast cancer recurrence [112] and are reassured by the “safety net” of continuing medical treatment and monitoring [9]. The MA.17 trial showed that extended adjuvant therapy with letrozole gives patients the opportunity of retaining the safety net for at least 5 more years after adjuvant tamoxifen [101], and these results led to its approval in this indication. Currently, letrozole is the only AI approved as an extended adjuvant therapy.
The final analysis of MA.17 after a median follow-up of 30 months showed letrozole significantly improved DFS (42% reduction in risk vs. placebo), distant DFS (40% reduction in risk vs. placebo) and, in node-positive patients, overall survival (39% reduction in risk vs. placebo) [101]. A recent cohort analysis of the MA.17 trial data suggested that the longer patients are exposed to letrozole, the greater the benefit, at least out to 48 months [59]. In addition, an extension to the MA.17 trial (MA.17 Re-randomization) is being conducted to determine the benefits of continuing letrozole for a further 5 years [102].
When a planned interim analysis of MA.17 revealed a significant advantage for letrozole, the trial was unblinded and patients on placebo were given the option to switch to letrozole [90]. In this non-randomized comparison, women from the placebo arm who elected to switch to letrozole also experienced an improvement in outcome when compared with those who elected to have no treatment [102].
Recurrence risk persists beyond the completion of adjuvant therapy, even in patients at low risk of recurrence. One study shows that with systemic adjuvant therapy, patients with node-negative breast cancer have a ≥25% 10-year risk of relapse and a corresponding 10-year breast cancer death rate as high as ≥10%, depending on tumor grade and size [113]. Thus, the proven efficacy of letrozole given after a tamoxifen-free period means that physicians need to discuss the option of restarting endocrine therapy with almost all patients. Physicians have to consider how best to address this topic with patients who are up to 2–3 years out beyond their initial 5 years of tamoxifen, i.e. about 5–8 years after their initial diagnosis. This will be a major communication challenge and create a dilemma for patients who may feel well and have put their breast cancer behind them.
Safety and compliance issues
The clinical benefits of AIs and tamoxifen are generally achieved without a major detrimental effect on overall QOL [114]. Data from the MA.17 trial, which is the only large adjuvant trial comparing AI therapy not with tamoxifen but with placebo, showed that overall QOL was maintained during extended therapy with letrozole, and only a minority of patients experienced substantial changes in QOL, which were compatible with a reduction in estrogen synthesis [115]. It is important to recognize that patients may attribute such changes to their treatment, whereas they could in fact be symptoms of menopause [116]. However, side effects do occur, which not only affect patient adherence to endocrine therapy [117] but can also lead to additional morbidity and even serious or life-threatening complications in a small minority of patients [51, 52, 90, 99, 108, 109].
The main safety concerns with long-term estrogen deprivation include potential effects on bone health, cardiac health, lipid profile, cognitive functioning, and sexual health [100]. The MA.17 trial showed that letrozole is well-tolerated in comparison with placebo [101], and BIG 1-98 showed that letrozole was better tolerated than tamoxifen [51] (Table 1). A recent patient-reported outcomes study in 104 tamoxifen-intolerant women found that switching to letrozole was associated with a significant reduction in hot flushes (P = 0.001) and significant improvements in QOL (P = 0.001) and mood (P = 0.04). Furthermore, when given the choice of continuing therapy, 66% of women indicated that they preferred to remain on letrozole, while only 24% preferred to go back to tamoxifen [118].
Despite an increase in newly diagnosed osteoporosis with AIs, no significant difference in clinical fracture rate was seen between letrozole and placebo [101]. A companion study to MA.17 showed that there was only a modest increase in bone resorption and reduction in bone mineral density (BMD) in the spine and hip with letrozole compared with placebo [119]. Consequently, patients should be made aware of the risk of osteoporosis and given advice on lifestyle measures (e.g. exercise, diet, vitamins) to reduce risk. A baseline BMD measurement should be obtained for all patients before starting therapy with an AI. BMD should then be measured annually, and patients at high risk of osteoporosis should be considered for prophylactic use of a bisphosphonate [120, 121]. However, recent data from ATAC may be reassuring for patients facing upfront AI therapy, since they indicate that women with normal initial BMD did not develop osteoporosis during a 5-year AI treatment [122].
Letrozole has been associated with an increase in arthralgia compared with placebo or tamoxifen, and myalgia compared with placebo [51, 101]. Arthralgia and joint pain can be bothersome and may lead to impaired mobility. Patients need to be advised that constant exercising of the joints will alleviate this adverse event and that use of nonsteroidal anti-inflammatory drugs over a certain period of time may be helpful. Moreover, these complaints seem to be most frequent in the beginning of AI therapy [123].
Another potential consequence of endocrine therapy is a detrimental effect on sexual health [115, 116]. Vaginal dryness is a consequence of menopause and treatments that cause menopausal symptoms, and has an important bearing on sexual health and well-being of breast cancer survivors [124]. Vaginal dryness can be a significant problem that can interfere with the stability of relationships [125], and patients need to receive counseling with regard to this issue [126]. It has been shown that clinical assessment and an active intervention program for menopausal symptom management in breast cancer survivors can lead to an improvement in sexual functioning [127]. A recent report demonstrated that the use of local estradiol therapy may cause elevation of serum estradiol and may therefore be contraindicated in postmenopausal women on AI therapy [128]. So far, such data do not exist for estradiol-containing compounds; thus, local estradiol treatment may be indicated in individual cases. Androgen treatment may also improve sexual well-being in postmenopausal women [129], yet the oncological safety of this approach has not been validated in patients receiving AI therapy. Of note, the incidence of vaginal dryness in the MA.17 trial was similar in the letrozole and placebo groups (Table 1) [101].
Differential effects on lipid profiles and cardiac risk have been reported between tamoxifen and AIs [51, 130, 131], but data from the Letrozole, Exemestane, and Anastrozole Pharmacodynamics trial, directly comparing the effects of anastrozole, letrozole, and exemestane on lipid profiles in healthy postmenopausal women, suggest that the steroidal and nonsteroidal inhibitors have a similar impact on lipid profiles [132]. On the basis of evidence from large randomized trials, when comparing letrozole with tamoxifen or placebo, it appears that letrozole does not have clinically relevant adverse effects on lipids or cardiac risk during long-term adjuvant therapy, although further assessment is warranted [51, 101, 133].
While letrozole is well-tolerated, and any side effects that do occur can be managed, it is essential that patients are motivated to stay on therapy. This can be a particular challenge in the extended adjuvant setting. Thus, as patients now face the prospect of receiving endocrine therapy for 10 years or more, it is important to consider the overall life-long benefits and risks from the individual’s perspective. A low burden of adverse events and maintenance of QOL is important in maintaining adherence to long-term treatment [60]. In the MA.17 trial, the rate of patients choosing to discontinue therapy during the first year was similar for letrozole (n = 256/2,575) and placebo (n = 254/2,582) [90]. Letrozole is ingested orally and can be safely and conveniently taken at home. Physician contact with oral endocrine therapy can be much less frequent than, for example, with orally active chemotherapy where regular blood tests, side-effect monitoring, and resulting dose modifications make frequent physician contact mandatory. Patients with breast cancer prefer oral cancer therapy providing that it does not compromise treatment efficacy [134].
Conclusions
Patients with breast cancer face bewildering choices at a time when they are experiencing highly stressful circumstances. Moreover, many of these women are not well-informed and thus are not able to take part in treatment decisions. The Gathering Information on Adjuvant Endocrine therapy initiative decided to delineate women’s knowledge and experience of adjuvant endocrine therapy. Results thus far indicate that only 22% of patients were fully or highly involved in the decision to start adjuvant endocrine therapy [135, 136]. Many of the women who took part in the survey were not satisfied with the degree to which they were involved in treatment decision-making, and women 60 years or older had the lowest levels of involvement [135, 136]. The results from this survey show that information provided to patients about adjuvant endocrine therapy is suboptimal and indicate the need for programs to raise patient awareness. The medical community has a responsibility to help patients understand their prospects for survival and make the right choices about treatment [24]. Doctor–patient discussions and programs to raise patient awareness will increase patients’ knowledge about the individualization of treatment and may increase the number of women who take an active role in treatment decisions.
Patients with hormone-responsive breast cancer should be offered the opportunity of receiving the most effective endocrine therapy. Clinical evidence suggests that postmenopausal women should receive an AI rather than tamoxifen as their first option if tumor characteristics and individual side-effect profiles support this choice. Letrozole has consistently demonstrated superiority over tamoxifen in the metastatic and adjuvant treatment settings [51, 74, 91, 101]. As a result of its innovative clinical trial program [51, 74, 101], letrozole is approved for use in postmenopausal women throughout the breast cancer care continuum [137]. From the patient’s perspective, the major benefits of letrozole are improving prospects for long-term survivorship (“cure”) in the adjuvant setting and delaying progression and the need for chemotherapy in the metastatic setting.
Endocrine therapy is very effective, has a generally favorable safety profile, and adds to the efficacy of chemotherapy. However, a major challenge for both physicians and patients is ensuring compliance with long-term daily therapy. This may be a particular problem in the extended adjuvant setting, where the patient may face the prospect of life-long therapy. As patients no longer feel sick, it is understandable that they may forget to take a dose and gradually lose interest in continuing with the treatment. Letrozole is well-tolerated and, as with all AIs, the majority of adverse events are secondary to the suppression of estrogen. The most important adverse events from the patient’s perspective are the “visible” ones, such as hot flushes, vaginal dryness, and arthralgias. To achieve optimal compliance, patients need to feel that physicians are taking their adverse events seriously and taking appropriate steps to alleviate any problems. Physicians who treat very few breast cancer patients may not have sufficient experience with AIs to satisfactorily manage individuals who are experiencing these adverse events.
Letrozole is likely to continue to play a major role in the management of breast cancer in all settings (see the paper in this supplement by Drs. Ellis and Ma, on Femara® and the future). Considering its efficacy and favorable side-effect profile, it is the logical choice for inclusion in new regimens, including combinations with novel agents. As the future unfolds, the management of breast cancer is set for further change, and it is essential that patients are informed and educated so that they can actively participate in treatment decisions and thus derive the most benefit from treatment advances.

patient
breast cancer
letrozole
aromatase inhibitors
adjuvant therapy
neoadjuvant therapy


Variations in the FTO gene are associated with severe obesity in the Japanese

Variations in the fat-mass and obesity-associated gene (FTO) are associated with the obesity phenotype in many Caucasian populations. This association with the obesity phenotype is not clear in the Japanese. To investigate the relationship between the FTO gene and obesity in the Japanese, we genotyped single nucleotide polymorphisms (SNPs) in the FTO genes from severely obese subjects [n = 927, body mass index (BMI) ≥ 30 kg/m2] and normal-weight control subjects (n = 1,527, BMI < 25 kg/m2). A case-control association analysis revealed that 15 SNPs, including rs9939609 and rs1121980, in a linkage disequilibrium (LD) block of approximately 50 kb demonstrated significant associations with obesity; rs1558902 was most significantly associated with obesity. P value in additive mode was 0.0000041, and odds ratio (OR) adjusted for age and gender was 1.41 [95% confidential interval (CI) = 1.22–1.62]. Obesity-associated phenotypes, which include the level of plasma glucose, hemoglobin A1c, total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, and blood pressure were not associated with the rs1558902 genotype. Thus, the SNPs in the FTO gene were found to be associated with obesity, i.e., severe obesity, in the Japanese.
Introduction
Obesity is the most common nutritional disorder in developed countries, and it is a major risk factor for hypertension, cardiovascular disease, and type 2 diabetes (Kopelman 2000; Wilson et al. 2003). Genetic and environmental factors contribute to obesity development (Maes et al. 1997; Barsh et al. 2000; Rankinen et al. 2006). Recent progress in single nucleotide polymorphism (SNP) genotyping techniques has enabled genome-wide association studies on common diseases (Herbert et al. 2006; Frayling et al. 2007; Scuteri et al. 2007; The Wellcome Trust Case Control Consortium 2007; Hinney et al. 2007). Using a large-scale case-control association study, we found that secretogranin III (SCG3) (Tanabe et al. 2007) and myotubularin-related protein 9 (MTMR9) (Yanagiya et al. 2007) are involved in susceptibility to the obesity phenotype. Genome-wide association studies have shown that the fat-mass and obesity-associated gene (FTO) is also associated with the obesity phenotype (Frayling et al. 2007; Scuteri et al. 2007; Hinney et al. 2007). This association was also found in many Caucasian and Hispanic American populations (Frayling et al. 2007; Scuteri et al. 2007; Dina et al. 2007; Field et al. 2007; Andreasen et al. 2008; Wåhlén et al. 2008; Peeters et al. 2008), whereas it was not found in the Chinese Han population (Li et al. 2008). Among Japanese, body mass index (BMI) was higher in subjects who had the A allele of rs9939609, similar to that observed in Caucasians; however, this finding was not significant (Horikoshi et al. 2007). Another group reported that rs9939609 was associated with BMI in the Japanese (Omori et al. 2008). Thus, the association of SNPs in the FTO gene with obesity in the Japanese remains controversial.
To investigate the relationship between the FTO gene and obesity in the Japanese, we performed a case-control association study using patients with severe adult obesity (BMI ≥ 30 kg/m2) and normal-weight controls (BMI < 25 kg/m2); we found that SNPs in intron 1 of the FTO gene were associated with severe adult obesity.
Materials and methods
Study subjects
The sample size for severely obese Japanese subjects (BMI ≥ 30 kg/m2) was 927 (male:female ratio 419:508, age 48.7 ± 14.2 years, BMI 34.2 ± 5.4 kg/m2), whereas that for Japanese normal weight controls (BMI < 25 kg/m2) was 1,527 (male:female ratio 685:842, age 48.1 ± 16.5 years, BMI 21.7 ± 2.1 kg/m2). The severely obese subjects were recruited from among outpatients of medical institutes. Patients with secondary obesity and obesity-related hereditary disorders were not included, and neither were patients with medication-induced obesity. The normal-weight controls were recruited from among subjects who had undergone a medical examination for screening of common diseases. Clinical features of the subjects are illustrated in Table 1. Additionally, 1,604 subjects were recruited (male:female ratio 803:801, age 48.7 ± 16.9 years, BMI 22.66 ± 3.16 kg/m2) from the Japanese general population. Each subject provided written informed consent, and the protocol was approved by the ethics committee of each institution and that of RIKEN.
Table 1Clinical characterization of obese and control subjectsObeseControlP valueGender (M/F)419/508658/842Age (year)49.1 ± 14.248.2 ± 16.50.049Body mass index (kg/m2)34.50 ± 5.3921.65 ± 2.08 <0.000001Glucose (mg/dl)129.2 ± 49.697.7 ± 23.9<0.000001HbA1c (%)6.5 ± 1.85.1 ± 0.6<0.000001Total cholesterol (mg/dl)209.9 ± 37.9201.2 ± 36.4<0.000001Triglycerides (mg/dl)153.2 ± 99.5104.0 ± 73.2<0.000001High-density lipoprotein cholesterol (mg/dl)53.1 ± 18.965.1 ± 15.7<0.000001Systolic blood pressure (mmHg)136.4 ± 18.1123.4 ± 17.8<0.000001Diastolic blood pressure (mmHg)83.8 ± 12.076.0 ± 11.1<0.000001P values were analyzed using Mann–Whitney U test. Data are mean ± standard deviation
DNA preparation and SNP genotyping
Genomic DNA was prepared from the blood sample of each subject by using the Genomix (Talent Srl, Trieste, Italy). We searched for dbSNPs with minor allele frequencies (MAF) > 0.10 in the FTO gene of Japanese people. We selected 90 SNPs and were able to construct Invader probes (Third Wave Technologies, Madison, WI) for them (Supplementary Table 1). SNPs were genotyped using Invader assays as described previously (Ohnishi et al. 2001; Takei et al. 2002). Nine SNPs (rs9937053, rs9939973, rs9940128, rs7193144, rs8043757, rs9923233, rs9926289, rs9939609, and rs9930506) reported in a previous genome-wide association study (Scuteri et al. 2007) were genotyped using TaqMan probes (C__29910458_10, C__11776771_10, C__29621384_10, C__29387650_10, C__29387665_10, C__29693738_10, C__30270568_10, C__30090620_10, and C__29819994_10; Applied Biosystems, Foster City, CA, USA).
Statistical analysis
Genotype or allele frequencies were compared between cases and controls in three different modes. In the first mode, i.e., the additive mode, χ2 test was performed according to Sladek et al. (Sladek et al. 2007). In the second mode, i.e., the minor allele recessive mode, frequencies of the homozygous genotype for the minor allele were compared using a 2 × 2 contingency table. In the third mode, i.e., the minor allele dominant mode, frequencies of the homozygous genotype for the major allele were compared using a 2 × 2 contingency table. A test of independence was performed using Pearson’s χ2 method. P values were corrected by Bonferroni adjustment and P < 0.00017 [0.05/99 (total SNP number)/3 (number of modes)] was considered significant. The odds ratio (OR) and 95% confidence interval (CI) were calculated by Woolf’s method. We coded genotypes as 0, 1, and 2, depending on the number of copies of the risk alleles. OR adjusted for age and gender was calculated using multiple logistic regression with genotypes, age, and gender as independent variables. Hardy–Weinberg equilibrium was assessed using the χ2 test (Nielsen et al. 1998). Haplotype blocks were determined using Haploview (Barrett et al. 2005). Simple comparison of the clinical data among the different genotypes was performed using one-way analysis of variance (ANOVA). Simple comparison of the clinical data between case and control groups was analyzed using Mann–Whitney U test. Difference in BMI between genotypes was analyzed using a multiple linear regression, with BMI as the dependent variable and genotype as the independent variable, and with gender and age as covariates for BMI. Statistical analyses were performed using StatView 5.0 (SAS Institute, Cary, NC, USA). Power was calculated by the Monte Carlo method.
Results
Case-control association studies
We searched for dbSNPs with MAF > 0.10 in the FTO gene. By using Invader and TaqMan assay, we successfully genotyped 99 SNPs spanning the FTO gene (Supplementary Table 1). Using these SNPs, we performed tests of independence between the phenotype and genotypes of obesity at each SNP by using severely obese subjects (BMI ≥ 30 kg/m2) and normal weight controls (BMI < 25 kg/m2). For each SNP, the lowest P value among the three different modes was selected as the minimum P value. All SNPs, including rs1421084, were in Hardy–Weinberg equilibrium (P > 0.01) (Supplementary Table 1).
The power of the test was calculated by Monte Carlo method with different MAFs and different effect sizes. Effect of the risk allele on penetrance was assumed to be multiplicative; i.e., the penetrances for three genotypes were assumed to be a, ar, and ar2, respectively, where a and r denote the lowest penetrance and genotype relative risk, respectively. Supplementary Table 2 shows the calculated values of the power of the test with different MAFs and different genotype relative risks (r). The lowest penetrance (a) was calculated for each gender by assuming the affection rates of 2.3% for men and 3.4% for women (Yoshiike et al. 2002). Genotype relative risk (r) was assumed to be the same for both genders. Supplementary Table 2 shows that the test has significant power at relative high risk allele frequency when genotype relative risk is >1.7.
As shown in Fig. 1 and Supplementary Table 1, 15 SNPs demonstrated significant associations with the obesity phenotype; the threshold of significance using Bonfferoni correction was P < 0.00017. These SNPs included rs9939609 (Frayling et al. 2007) and rs1121980 (Hinney et al. 2007) that were reported to be significantly associated with the obesity phenotype in the Caucasian population, as determined by genome-wide association studies; rs9930506 (Scuteri et al. 2007) showed marginal association with obesity in the Japanese. Linkage disequilibrium (LD) analysis revealed that these 15 SNPs were in almost complete LD (D' > 0.98, r2 > 0.80) and were located within the same LD block of approximately 50 kb (Fig. 1). The most significant association was observed for rs1558902 [additive mode, P = 0.0000041 and allele-specific OR (95% CI) adjusted for age and gender was 1.41 (1.22–1.62)]. The minor alleles of rs9939609 (MAF = 0.24) and rs1121980 (MAF = 0.26) were significantly more frequent in the obese group than in the normal-weight control group (additive mode, P = 0.000012 and P = 0.000051, respectively), and ORs were 1.38 (95% CI = 1.20–1.59) and 1.33 (95% CI = 1.16–1.52), respectively (Table 2, Supplementary Table 1). The MAF of both SNPs in the control group was 0.18; this was consistent with data obtained from the haplotype map of the human genome (HapMap) (Supplementary Table 1). Our data indicated that the SNPs in the FTO gene were associated with severe obesity in the Japanese.
Fig. 1Linkage disequilibrium (LD) mapping, polymorphisms, and P values obtained in the test of independence between the phenotype and genotypes of obesity at various single nucleotide polymorphisms (SNPs) in the fat-mass and obesity-associated gene (FTO) gene. P values are expressed as negative logarithm of the minimum P values obtained in the three models (additive, minor allele dominant, and minor allele recessive modes). LD coefficients (D') between each pair of SNPs were calculated and are displayed as a strand in the LD blocks. Minor allele frequencies of all SNPs used in this analysis are ≥10%. The genomic structure is shown in the upper. The gray bar marks the LD block associated with obesityTable 2Associations of single nucleotide polymorphisms (SNPs) in the fat-mass and obesity-associated gene (FTO) gene with obesity existing in the 50-kb linkage disequilibrium (LD) blockdbSNP IDAlleleGenotypeAdditive modeRecessive modeDominant modeCaseControl1/2111222Sum111222SumOR (95% CI)χ2P valueχ2P valueOR (95% CI)χ2P valueOR (95% CI)rs9937053A/G593604949136341477312501.31 (1.13–1.51)12.30.00047 2.00.161.30 (0.90–1.88)13.00.000311.37 (1.16–1.63)rs9939973A/G613674969247550494115201.32 (1.15–1.51)15.70.000077a3.00.0811.36 (0.96–1.93)16.10.000061a1.40 (1.19–1.66)rs9940128A/G603664989247550094115161.31 (1.15–1.50)15.20.00010a2.60.111.33 (0.94–1.89)15.90.000068a1.40 (1.19–1.65)rs1421085C/T4933853792457443101915191.38 (1.20–1.59)19.60.000011a3.30.0681.44 (0.97–2.12)20.00.0000078a1.47 (1.24–1.74)rs1558902A/T4834153692552449102115221.41 (1.22 -1.62)21.20.0000041a4.60.0321.55 (1.04–2.31)20.80.0000052a1.48 (1.25–1.75)rs1121980A/G613674999277350494715241.33 (1.16–1.52)16.50.000051a3.60.0591.40 (0.99–1.99)16.50.000050a1.41 (1.19–1.66)rs7193144C/T4933953292055447101415161.39 (1.21–1.61)20.40.0000067a4.00.0441.49 (1.01–2.22)20.30.0000067a1.47 (1.24–1.74)rs8043757T/A4831954190854436102715171.36 (1.18–1.57)17.40.000037a4.20.0401.51 (1.02–2.25)16.40.000052a1.42 (1.20–1.69)rs8050136A/C5133653892556450101815241.38 (1.20–1.59)19.40.000012a4.70.0311.53 (1.04–2.26)18.50.000017a1.45 (1.22–1.71)rs3751812T/G5134053492555458101315261.38 (1.20–1.59)19.60.0000098a5.10.0241.56 (1.06–2.31)18.50.000017a1.45 (1.22–1.71)rs9923233C/G5133553391955449101015141.38 (1.20–1.60)19.80.0000093a5.00.0251.56 (1.06–2.30)18.70.000015a1.45 (1.23–1.72)rs9926289A/G503235319045642599314741.37 (1.19 -1.58)18.70.000020a3.90.0471.48 (1.00–2.19)18.10.000021a1.45 (1.22–1.72)rs9939609A/T5133453491956443100515041.38 (1.20–1.59)19.50.000012a4.50.0341.52 (1.03–2.24)18.70.000015a1.45 (1.23–1.72)rs7185735G/A5134053692755455101415241.38 (1.20–1.59)19.90.0000089a5.00.0251.55 (1.05–2.30)18.80.000014a1.45 (1.23–1.72)rs9931494G/C643634949217150494215171.35 (1.18–1.55)18.40.000018a5.60.0181.52 (1.07–2.15)16.90.000039a1.42 (1.20–1.67)rs17817964T/C623615009236852493015221.30 (1.14–1.49)13.50.00022 5.80.0161.54 (1.08–2.19)11.40.000751.33 (1.13–1.57)rs9930506G/A673654889208252191315161.28 (1.12–1.46)12.80.00038 3.50.0611.37 (0.98–1.92)12.10.000511.34 (1.14–1.58)rs9932754C/T663684919257852591915221.29 (1.13–1.48)13.60.00023 4.20.0401.42 (1.01–2.00)12.60.000401.35 (1.14–1.59)rs9922619T/G663684899237852991915261.29 (1.13–1.48)13.50.00024 4.30.0381.43 (1.02–2.01)12.30.000441.34 (1.14–1.58)rs7204609C/T13441837392527371752915190.83 (0.73–0.93)9.680.0022 5.00.0250.77 (0.62–0.97)7.50.00630.79 (0.67–0.94)rs12149832A/G533495259276248098215241.33 (1.15–1.53)15.20.000098a3.50.0611.43 (0.98–2.08)14.80.00012a1.39 (1.17–1.64)The odds ratio (OR) for each SNP was adjusted simultaneously for age and gender using additive modelCI confidence interval, χ2 chi-squareaSignificant P value (P < 0.00017)
Analysis of various quantitative phenotypes with rs1558902
To investigate whether the genotypes of SNP rs1558902 are associated with the phenotypes of metabolic disorders, we compared the following among the different genotypes in the cases, controls, and combined groups: ANOVA results, BMI, levels of fasting plasma glucose, hemoglobin A1c (HbA1c), total cholesterol, triglycerides, HDL cholesterol, and blood pressure. As rs1558902 showed the most significant association with obesity and its call rate was the highest, we analyzed various quantitative phenotypes by using this SNP. The quantitative phenotypes regarding BMI and the levels of fasting plasma glucose, HbA1c, total cholesterol, triglycerides, HDL cholesterol, and blood pressure were not found to be significantly associated with the genotypes at rs1558902 in either the case or control group (Table 3). Although there was no significant difference in BMI values among genotypes in either the control or case group, the direction of the difference (AA > AT > TT) was in accordance with the association between the qualitative obesity phenotype and the genotype shown.
Table 3Comparison of various quantitative phenotypes among different genotypes at single nucleotide polymorphism (SNP) rs1558902 in obese and control subjectsObeseControlAA (n = 48)AT (n = 341)TT (n = 536)AA (n = 52)AT (n = 448)TT (n = 1022)Age (year)49.8 ± 15.349.6 ± 14.348.8 ± 14.146.9 ± 15.446.9 ± 16.748.8 ± 16.5P value0.640.098BMI (kg/m2)35.16 ± 5.7034.61 ± 5.4334.39 ± 5.3321.94 ± 2.23 21.62 ± 2.1021.65 ± 2.06P value0.580.56Glucose (mg/dl)142.8 ± 54.8125.4 ± 43.2130.8 ± 53.3101.7 ± 44.196.3 ± 18.198.2 ± 24.7P value0.0540.34HbA1c (%)6.9 ± 2.16.4 ± 1.76.5 ± 1.85.1 ± 1.25.0 ± 0.55.1 ± 0.7P value0.190.15Total cholesterol (mg/dl)215.1 ± 46.7211.3 ± 38.8208.6 ± 36.6195.6 ± 38.8201.4 ± 37.8201.4 ± 35.6P value0.370.53Triglycerides (mg/dl)171.7 ± 119.5151.3 ± 102.1153.2 ± 96.0111.7 ± 70.6102.0 ± 71.4104.4 ± 74.2P value0.420.63HDL cholesterol (mg/dl)53.2 ± 13.854.8 ± 24.052.0 ± 15.462.1 ± 14.265.1 ± 15.965.3 ± 15.6P value0.140.53SBP (mmHg)134.2 ± 20.4137.0 ± 17.8136.2 ± 18.2122.7 ± 17.3123.2 ± 18.8123.5 ± 17.5P value0.610.91DBP (mmHg)80.3 ± 11.784.1 ± 12.083.9 ± 12.075.5 ± 11.175.2 ± 11.776.3 ± 10.9P value0.140.22Data of each quantitative phenotype were compared among different genotypes at the rs1558902 in obese and control subjects. P values were analyzed using analysis of variance in each group of obese and control subjects. Data are mean ± standard deviation.HDL high-density lipoprotein, SBP systolic blood pressure, DBP diastolic blood pressure.
Finally, we examined the BMI distribution of rs1558902 in the Japanese general population and found that rs1558902 genotype was significantly associated with BMI (Table 4). This result would confirm the association of rs1558902 with obesity.
Table 4Association of body mass index (BMI) with rs1558902 genotypes in the Japanese general populationAAATTTP value (additive model)aBMI (kg/m2) (n)23.17 ± 3.20 (59)22.79 ± 3.26 (482)22.57 ± 3.11 (1063)0.041aThe difference in BMI according to genotypes was analyzed using a multiple linear regression, with BMI as the dependent variable and genotype as the independent variable and with gender and age as covariates for BMI. Data are represented as mean ± standard deviation
Discussion
Recent genome-wide association studies have shown that the FTO gene is associated with obesity (Frayling et al. 2007; Scuteri et al. 2007; Hinney et al. 2007). The associations between variations in the FTO gene and the obesity phenotype have been observed in many Caucasian subjects (Frayling et al. 2007; Scuteri et al. 2007; Dina et al. 2007; Field et al. 2007; Andreasen et al. 2008; Wåhlén et al. 2008; Peeters et al. 2008). However, these associations were controversial with regard to Asian subjects (Horikoshi et al. 2007; Li et al. 2008; Omori et al. 2008). BMI values did not significantly differ among the genotypes in the general population of Chinese and Japanese (Horikoshi et al. 2007; Li et al. 2008). We performed a case-control association study with regard to severe obesity and found that the SNPs in the FTO gene were significantly associated with severe obesity. Although the SNPs demonstrated the most significant association in the Japanese, which was different from that in Caucasians, the significantly associated SNPs existed in a similar block as that in Caucasians. Therefore, the FTO gene could also contribute to the development of severe obesity in the Japanese.
BMI was modestly different among rs1558902 genotypes in the general population in this study; rs9939609 was not significantly associated with BMI in the general population (AA 23.22 ± 3.14 vs AT 22.79 ± 3.25 vs TT 22.58 ± 3.13, P = 0.063). In the Japanese population, rs1558902 may be more tightly associated with BMI than rs9939609. The National Nutrition Survey of Japan reported that the prevalence of subjects with a BMI of ≥30 kg/m2 is only 2.3% in men and 3.4% in women aged 20 years and older (Yoshiike et al. 2002), and the mean BMI was approximately 23 kg/m2 for ages 15–84 years (Yoshiike et al. 1998). Inconsistency in the results of effects of variations in the FTO gene on BMI between Japanese and Europians may be due to the relatively small mean and variance of BMI in the former than the latter.
The significant SNPs were located in intron 1 of the FTO gene. The rs1558902 and other significant SNPs, for example, rs9939609 and rs1121980, would affect transcriptional activity of the FTO gene, although further investigation is necessary. The precise mechanism by which the FTO gene leads to obesity development is unclear (Gerken et al. 2007; Sanchez-Pulido et al. 2007). However, the FTO gene is expressed in the hypothalamus and regulated by fasting and leptin (Frayling et al. 2007; Gerken et al. 2007). Using large-scale case-control association studies, we determined that the SCG3 (Tanabe et al. 2007) and MTMR9 (Yanagiya et al. 2007) genes are involved in susceptibility to the obesity phenotype. These two genes are expressed in the hypothalamus. Genetic studies in mice have suggested that mutations in several genes, such as those encoding leptin, proopiomelanocortin, and melanocortin-4 receptor, are implicated in a monogenic form of inherited obesity (Barsh et al. 2000; Rankinen et al. 2006). Such mutations have also been reported in obese humans. As most such genes are expressed in the hypothalamus and have been indicated to play important roles in the regulation of food intake, genes expressed in the hypothalamus are likely to be good candidates for susceptibility to obesity.
In summary, we have identified the genetic variations in the FTO gene that may influence the risk of severe obesity in the Japanese.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Summary of the association of SNPs between cases and controls (DOC 315 kb)

association
obesity
fat-mass and obesity-associated gene
snp
japanese population


Cisplatin-DNA adduct formation in patients treated with cisplatin-based chemoradiation: lack of correlation between normal tissues and primary tumor

Purpose In this study, the formation of cisplatin-DNA adducts after concurrent cisplatin-radiation and the relationship between adduct-formation in primary tumor tissue and normal tissue were investigated.
Introduction
Concurrent chemoradiotherapy is more effective than radiotherapy (RT) alone, both in in vitro studies [1, 2] as well as in clinical studies in many different tumor types, including advanced head and neck squamous cell carcinoma (HNSCC) and cervical cancer, leading to improvements in locoregional control and/or survival [11, 13, 21, 28]. In a metaanalysis on concurrent chemoradiation in HNSCC, the addition of concurrent single agent cisplatin to RT was the most effective treatment regime with the largest improvement on overall survival [5]. Concurrent cisplatin-based chemoradiation is now considered standard care in advanced-stage HNSCC and cervical cancer.
In addition to the increased efficacy of the combined treatment, it was shown that the concurrent regimens are accompanied by higher acute toxicity rates compared to radiation alone [11, 21], with more severe mucositis and gastrointestinal toxicity.
Since a substantial number of patients treated with concurrent chemoradiation still fail to respond to this toxic treatment, there is a need for an accurate predictive assay, based on which patients that are likely to respond to the therapy can be selected. This strategy may also provide a tool to individualize and tailor treatment, based on evaluation of the predictive assay, early during therapy.
One potential predictive marker is the formation of cisplatin-DNA adducts, which are formed when cisplatin reacts with the cellular DNA by binding to nucleotides. The majority of adducts are either intrastrand adducts with cisplatin bound between two guanosine (GG) nucleotides or adenosine–guanosine (AG) nucleotides [8]. Cisplatin-DNA adducts can be measured in tumor and normal tissue. The level of adducts has been shown to correlate with cytotoxicity in vitro [34], and with response to therapy in patients [3, 17, 32, 35]. In most of these studies [3, 32, 35], adduct measurements were performed in the normal tissue, with the assumption that normal tissue can be used as surrogate marker for tumor.
In our institute, study protocols with cisplatin-DNA adduct measurements are ongoing in patients with HNSCC and cervical cancer, who are all treated with concurrent cisplatin-based chemoradiation. The objectives of the current study are (1) to investigate the two major forms of cisplatin-DNA adducts (GG and AG adducts) after different schedules of cisplatin given concurrently with radiation and (2) to explore relationships between adducts in primary tumor and normal tissue. We specifically wanted to investigate whether the level of adducts in tumors are reflected by those in normal tissues. In studies focused on the predictive value of cisplatin-DNA adduct levels, this would then justify the use of more easily obtained normal tissues as a surrogate for tumor samples.
Patients and methods
Concurrent chemoradiation protocols
This study on adduct formation was approved by the medical ethical committee of the participating hospitals. The main eligibility criteria were: patients scheduled for cisplatin chemoradiation, no previous treatment with cisplatin, and informed consent. Eligible patients were informed about the nature of the protocol and after written informed consent they were entered in the study. Patients were recruited from one of the following regimens.
In advanced-stage HNSCC patients, two different cisplatin-based concurrent chemoradiation protocols (RADPLAT) were used. The RADPLAT 100 schedule, which is the most commonly administered schedule in HNSCC [11], consisted of cisplatin given intravenously (IV) at a dose of 100 mg/m2, as a 30 min infusion, 1–2 h before RT at days 1, 22, and 43 of treatment. This treatment was part of a randomized trial on IV vs. intra-arterial chemoradiation. In RADPLAT daily LD, low-dose (LD) cisplatin was given as a 1–2 min IV infusion at a dose of 6 mg/m2 daily, for a total number of 20 doses, 1–2 h prior to RT. This treatment was shown to be an effective alternative in HNSCC [6, 19]. Patients ineligible for or refusing the randomized trial on intra-arterial chemoradiation were treated with RADPLAT daily LD, since this treatment could be given on an outpatient basis. The RT target volumes for all schedules included the primary tumor and the bilateral neck at a dose of 46 Gy in 23 fractions. A boost was given to the macroscopic tumor extensions at the primary tumor site and lymph node metastases at a dose of 24 Gy in 12 fractions, resulting in a total dose of 70 Gy in 35 fractions.
In patients with advanced-stage squamous cell cervical cancer, concurrent chemoradiation (CERVIX 40) consisted of weekly administration of cisplatin IV as a 4-h infusion at a dose of 40 mg/m2, followed by RT within 1–2 h. The total number of doses was 5–6, depending on external beam RT schedule and the number of intracavitary brachytherapy applications. The total radiation dose was usually 46 Gy to the cervical tumor, uterus, and pelvic lymph nodes, with a boost to the cervix tumor and other involved regions, to a total dose of 60–74 Gy, depending on treated volume and whether or not intracavitary brachytherapy was given.
Cisplatin-DNA adducts
Before and after chemotherapy, normal tissue samples [white blood cells (WBC) and buccal cells] were collected. In patients with an accessible primary tumor, a biopsy of the tumor was also taken. To avoid harvesting necrotic tissue, the biopsy was taken at the viable peripheral rim of the tumor. Samples were obtained at different times, due to logistic reasons: for the patients in the RADPLAT 100 study, this was done 23 h after the end of administration of the first cisplatin infusion (given on day 1 of treatment). In the patients in the CERVIX 40 study, samples were taken 20 h after the end of administration of the first weekly cisplatin infusion (given on day 1 of treatment). For the patients in the RADPLAT daily LD group, samples were taken 1 h after the 5th dose on day 5 of treatment. WBC were isolated from whole blood samples according to a previously published protocol [24]. Buccal cells were collected in phosphate-buffered saline by scraping the bilateral buccal mucosa using a cotton swab. In HNSCC patients, the buccal mucosa could be located within the RT treatment fields, depending on the tumor site. The harvested cells were centrifuged (5 min at 4°C, 1,000 rpm) and resuspended in a Tris–EDTA buffer and stored at −80°C until analysis. Tumor biopsies were taken and immediately frozen at −80°C until analysis. Quantification of GG- and AG-intrastrand adducts was performed by a 32P-postlabeling technique as previously described [29]. Internal standardization was incorporated in the present analysis method, by adding 300 fmol of TT nucleotides to each sample. From previous work, the reproducibility of the assay is known by analysis of duplicate specimens within the same experiment (within-run reproducibility) and by analysis of duplicate specimens in separate experiments (between-run reproducibility) [29]. The reproducibility was described for WBC and tumor samples and was within 10% for the within-run precision and between 2–20% for the between-run precision. It was also determined for buccal cells in the same way and similar reproducibility was obtained. The concentration of DNA present in the samples was measured spectrophotometrically at 260 nm with the Nanodrop ND-1000 (Nanodrop Technologies Inc, Wilmington, DE, USA). The cisplatin-DNA adduct levels were expressed as fmol/μg DNA. The lower limit of quantification for the Pt-GG and Pt-AG adducts was 0.087 and 0.053 fmol/μg DNA, respectively.
Statistical analysis
Analysis of data was performed in SPSS software (version 11.5, SPSS, Inc.). For quantitative comparison of numerical data between groups, the Student’s t-test was applied. The Pearson correlation coefficient and Spearman’s rank correlation coefficient were calculated for analysis of correlations between different samples (WBC, buccal cells, and primary tumor) on an intra-patient level.
Results
Samples for cisplatin-DNA adduct determination were obtained from 63 patients: 27 from RADPLAT daily LD, 15 from CERVIX 40, and 21 from RADPLAT 100. WBC samples were taken from 61 patients, buccal cells from 25, and tumor biopsies from 23 of these patients. The reasons for the missing data for the normal tissue samples were: no collection of samples due to logistics or (in minority of cases) not sufficient volume for analysis. The reason for missing primary tumor biopsy data were: tumor not accessible for direct outpatient-based biopsy (in HNSCC patients) or refusal (in cervix cancer patients).
In WBC, all but three of the 60 available baseline samples were below the LLQ for the GG adducts and all but two below the LLQ for the AG adducts. This was probably due to some background signal inherent in the postlabeling method, since all patients had not been treated before with platinum chemotherapy. The yield of DNA, obtained from the buccal cell samples was rather low, ranging from 1–10 μg. Baseline samples of buccal cells were available from 16 of 25 patients, of whom posttreatment samples were also available. The baseline values of GG adducts in buccal cells ranged from 0.067 to 0.745 fmol/μg DNA (mean 0.282, SD 0.19) and baseline values of AG adducts in buccal the cells ranged from 0.087 to 1.538 fmol/μg DNA (mean 0.398, SD 0.38). All but two of the baseline GG-adduct values were above the LLQ and all the baseline AG-adduct values were above the LLQ. This was probably due to the low DNA quantities obtained from the buccal cell samples. The difference in adduct levels from baseline to post-infusion values was significant for the GG adducts, but not for the AG adducts. This implies that for measuring low quantities of AG adducts in the low amounts of buccal cell DNA available, we reached the limits of quantification with this postlabeling method.
In Table 1, the results are presented for the GG- and AG-adduct levels in normal tissue and primary tumor for the three different cisplatin-chemoradiation regimes. Adduct levels in primary tumor were two to five times higher than those in WBC for all three treatment regimes for both GG- and AG-adduct formation (Student t-test, P < 0.001 for both adduct types). For the comparison of the adduct levels from the three different treatment protocols, the data from the RADPLAT daily LD were omitted, since the daily administration schedule and sampling time (1 h after the infusion) were different from the other two regimens. The adduct levels in the RADPLAT 100 schedule were statistically significantly higher than those after the CERVIX 40 schedule for both tumors (Student t-test, P = 0.01) and normal tissues (Student t-test, P < 0.01).
Table 1Cisplatin-DNA adducts (in fmol/μg DNA) in normal tissue and primary tumor after different schedules of cisplatin-based chemoradiationTreatment scheduleWBCBuccal cellsTumorGGAGGGAGGGAGRADPLAT daily LDN2626111166 Mean0.340.050.840.210.660.10 SD0.100.190.390.140.370.05CERVIX 40N1414771010 Mean0.440.060.870.221.940.26 SD0.170.030.260.061.470.26RADPLAT 100N21217777 Mean1.0470.1241.5630.3403.8660.413 SD0.3770.0490.4340.0901.1010.089See text for explanation of treatment schedulesN number of patients, SD standard deviation, WBC white blood cells, GG GG-adducts, AG AG adducts
A highly significant linear correlation (Pearson correlation, r = 0.93, P <0.001, n = 61) was observed between the level of GG and AG adducts in WBC (see Fig. 1a), with a mean ratio of GG/AG adducts of 7.6 ± 2.1 SD. Similar linear relationships and ratios were found for GG and AG adducts in primary tumor (r = 0.89, P < 0.001, n = 23, and ratio 8.5 ± 2.8; Fig. 1b) and buccal cells (r = 0.85, P < 0.001, n = 23, and ratio 3.9 ± 1.3; Fig. 1c).
Fig. 1Correlation-plots of GG- and AG-adduct levels in white blood cells (WBC) (panel a), primary tumor biopsy (panel b), buccal cells (panel c). In each panel, the three different treatment groups are depicted: RADPLAT daily LD 5 × 6 mg (red squares), CERVIX 40 mg (green circles), and RADPLAT 100 mg (blue triangles)
A trend was observed between GG-adduct levels in WBC and buccal cells, although not significant (r = 0.38, P = 0.07, n = 24). No significant correlations were found between tumor and normal tissue: tumor vs. WBC (r = 0.35, P = 0.13, n = 21) and tumor vs. buccal cells (r = −0.003, P = 0.99, n = 9). See Fig. 2 for scatter plots. Similar results were found for the AG adducts: no significant correlations were found between adducts in tumor vs. WBC (r = 0.14, P = 0.55, n = 21), tumor vs. buccal cells (r = 0.25, P = 0.58, n = 7) or WBC vs. buccal cells (r = 0.26, P = 0.24, n = 22).
Fig. 2Correlation plots of GG adducts in white blood cells (WBC) vs. buccal cells (panel a), and normal tissue vs. tumor (panels b and c). In each panel, the three different treatment groups are depicted: RADPLAT daily LD 5 × 6 mg (red squares), CERVIX 40 mg (green circles), and RADPLAT 100 mg (blue triangles)
Discussion
There are two main conclusions from the 63 patients included in these analyses. First, intra-tumoral adduct levels were substantially higher than those in normal tissue (WBC) at all cisplatin-dose levels examined. Second, no positive correlations were evident between adducts in tumors and normal tissues. It should be noted that the various schedules, the cisplatin doses, and the duration of infusions differed, as well as the sampling times. However, all analyses on adducts were performed on paired samples, within the same patient. This eliminates variance of these factors since the normal tissue and tumor samples all were obtained at similar time points after the cisplatin infusion. In the RADPLAT daily LD, some accumulation from the previous four daily 6 mg cisplatin infusions would have occurred and affected the day 5 measurement after the 5th infusion. In the RADPLAT 100 and CERVIX 40 patients, no such accumulation would have occurred, since the sampling was done 20–23 h after the first infusion of cisplatin.
Relatively little information is available regarding the in vivo formation of intra-tumoral cisplatin-DNA adducts in clinical series [17, 23]. Most studies focused on intra-tumoral platinum concentrations, both in HNSCC [12, 33] and cervical cancer patients [15, 22]. These studies are mostly characterized by relatively low numbers of patients, probably due to the invasive nature of the procedure. The data on correlations between adducts and platinum content are contradictory: In an experimental study [37], no relationship could be established between the intra-tumoral adduct levels and platinum content, although in one clinical study, a significant correlation was found [23].
Adduct levels in primary tumors were consistently two- to fivefold higher than in WBC. This was true for both GG and AG adducts. This finding was previously described in anecdotal clinical cases [9, 30]. Similar observations were made in platinum content studies in an experimental tumor model [18] and in HNSCC patients [33]. Adducts in tumor were also higher than in buccal cells in the CERVIX 40 and RADPLAT 100 group, but not in the RADPLAT daily LD group. We observed a linear relationship between the two major adduct forms (AG and GG) for both normal tissues and tumor (Fig. 1a–c), although GG-adduct formation was 5–12 times increased relative to the AG adducts, as reported earlier [8, 36]. The linear relationships between GG and AG adducts serve as a validation of the assay. Apparently within a sample, both types of adducts are present in an equal proportion, although the absolute amounts differ greatly. In previous studies, the type of adduct responsible for the cytotoxic effect of platinum compounds has been investigated. From two of these studies [7, 36] it was concluded that the AG adduct was responsible for the platinum cytotoxicity. From our study, such a conclusion cannot be made, since both types of adducts were present in equal proportions.
Adduct formation in different tissue samples showed a lack of correlation between tumor and normal tissue. One might have expected that higher adduct levels in normal tissue would be accompanied by more adducts in tumor, although this was not the case. Similar results were found in an animal study [25] and in a clinical series of uterine cervix cancer patients [22], both demonstrating lack of correlation between tumor platinum and serum platinum concentrations. If adduct formation were merely a matter of cisplatin exposure, then a positive correlation would be expected. The reasons for the higher levels of adducts in tumor vs. normal tissue and the lack of correlation between them may be explained by differences between tumor and normal tissue in one of the following factors: Tumors are heterogeneous in terms of blood supply and perfusion, resulting in differences in cisplatin uptake and diffusion, drug-pumps may diminish intra-cellular cisplatin concentrations by active transmembrane transport of cisplatin, prohibiting adducts to be formed, and tumor cells may have less effective capacities to repair damage from cytotoxic agents.
Adducts are formed rapidly and in a dose-dependent fashion within 1–2 h after cisplatin exposure, with a gradual decrease (repair) within the next 20–24 h [20, 32, 36]. The persistence of cisplatin-DNA adducts may therefore be regarded as a measure of repair and possibly be used as predictive assay. We therefore chose to measure adducts 20–23 h after the end of chemotherapy infusion. Ideally, more frequent measurements would have generated more information on the rate of adduct formation, repair, and total exposure to adducts (like an AUC analysis) [24]. However, obtaining repeated biopsies is not feasible in clinical practice.
The results from the buccal cell samples need to be interpreted with caution, especially the AG-adduct levels, since uncertainties remain. With the low quantities of AG-adducts in the low amounts of buccal cell DNA we could extract, we reached the limits of quantification of the postlabeling method.
The rationale for measuring cisplatin-DNA adducts is that it could be used as a predictive assay: higher levels of adducts would predict favorable treatment outcome. Many studies have been performed for this purpose, investigating adducts in normal tissue (WBC and buccal cells) [3, 4, 10, 14, 26, 31, 32, 35]. These study designs are based on the assumption that normal tissue can be used as a surrogate marker for tumor tissue with respect to cisplatin-DNA adduct formation. In our present study, however, we did not find such a correlation. This might explain why the results on the role of adduct formation to predictive outcome are heterogeneous and contradictory, as illustrated below.
Several studies on adduct formation in WBC showed that the level of adducts was positively correlated with response to chemotherapy in patients with advanced disease in a variety of tumor sites [31, 32], while others showed no correlation [4, 26]. One study showed a positive correlation in one tumor site (ovarian cancer), but not in the other (breast cancer) [14], and in another study the level of adduct formation showed a negative association with survival for day-5 adducts, while there was no difference for day-1 adducts [10]. In studies on adduct levels in buccal cells, a positive correlation was found between adducts and either disease response [3] or better survival in non-small cell lung cancer (NSCLC) [35]. We recently showed that adduct formation in primary tumor appeared to be associated with better progression-free survival in HNSCC [17].
Differences were observed between the levels of intra-tumoral adducts for the three chemoradiation schedules, with lower adducts after lower dosages of cisplatin. Based on this, however, one cannot predict that the schedules with less adducts will result in less cytotoxicity, since not only the cisplatin dose, but also timing and schedule of cisplatin administration are crucial determinants of efficacy [1]. These factors may contribute to differences in the formation and rate of repair of adducts, resulting in different exposures to cisplatin-DNA adducts. These differences make it difficult to extrapolate from the observed adduct values in tumor and normal tissues to a prediction of superiority of one schedule over another.
In future studies, immunohistochemistry on repair proteins like ERCC1 [27] or gene expression profiling studies on platinum resistance [16] could be used to improve the prediction of cisplatin sensitivity and prediction of therapy response.
In conclusion, we have demonstrated that in concurrent chemoradiotherapy schedules, cisplatin adduct levels in tumors were significantly higher than in normal tissues (WBC). No evidence of a correlation was found between adduct levels in normal tissues and primary tumor biopsies. This lack of correlation may, to some extent, explain the inconsistencies in the literature regarding whether or not cisplatin-DNA adducts can be used as predictive test in anticancer therapy.
Conflict of interest: none declared.

cisplatin
chemoradiation
cervical cancer
dna adducts
head and neck cancer