Treatment Strategies Using Letrozole andTamoxifen in a Xenograft Model for BreastCancerCombined Treatment versus Alternating or Sequential Treatment

Angela H. Brodie, Danijela Jelovac and Brian LongDepartment of Pharmacology and Experimental Therapeutics, School of Medicine, University of Maryland, Baltimore,Maryland, USA

Abstract A tumor xenograft model has been established that more closely relates to hormone-dependent breast cancerthan tumors induced with the carcinogens in the rat. The MCF-7Ca tumors are developed from MCF-7 cellstransfected with aromatase. These cells synthesize estrogens and are responsive to the effects of estrogen viaestrogen receptors. Tumor growth is inhibited by both aromatase inhibitors and antiestrogens. The purpose ofthese studies was to determine the effectiveness of several strategies of treatment with aromatase inhibitors andantiestrogens. Experiments have shown that tumor growth can be inhibited more effectively with aromataseinhibitors such as letrozole than with tamoxifen. Furthermore, the model demonstrated that an aromatase inhib-itor alone is more effective than in combination with tamoxifen administered either together or sequentially.This result predicted the outcome of such clinical trials as ATAC (Arimidex and Tamoxifen Alone or inCombination) and suggests that the model is a good predictor of responses observed in human breast cancerpatients. Thus, the model could be useful to guide the design of future clinical trials in patients with hormone-dependent breast cancer.

REVIEW ARTICLE Am J Cancer 2003; 2 Suppl. 1: 1-61175-6357/03/0001-0001/$30.00/0

© Adis International Limited. All rights reserved.

Previous studies of aromatase inhibitors were carried out incycling rats with mammary tumors chemically induced with suchagents as 7,12-dimethylbenzanthracene (DMBA) or N-methyl-N-nitroso-urea (MNU).[1] Although these tumors are useful for dem-onstrating the efficiency of aromatase inhibitors, their formationis dependent on ovarian estrogen synthesis. Therefore, the modelwas not relevant in postmenopausal women, the patient popula-tion most likely to benefit from these agents.

After menopause, ovarian production of estrogens ceases.Estrogen synthesis is limited to non-ovarian tissues, such as adi-pose tissue, and is no longer under gonadotropin control. A sig-nificant amount of estrogen is produced in breast tissue, such thatbreast estrogen levels in postmenopausal women are reported tobe 4–6 times higher than circulating levels, and comparable tothose found in premenopausal breast tissue.[2] Estrogen receptor(ER) concentrations also increase in the breast with age. Thus, alarge proportion of postmenopausal patients have ER-positivetumors and are responsive to ‘hormonal’ manipulation. To mimicaspects of estrogen-dependent breast cancer biology in the

postmenopausal patient, a xenograft model was developed basedon utilizing the ER-positive human breast cancer cell line stablytransfected with aromatase to provide a source of estrogen inovariectomized mice.[3,4] This article reviews the development ofthe model and past and current data pertaining to the effect ofantiestrogens and aromatase inhibitors on tumor growth. In addi-tion, the value of the model to the design of clinical trials isdiscussed.

1. Intratumoral Aromatase Model of Breast Cancer

The antiestrogen tamoxifen has proved to be of significantbenefit in treatment of postmenopausal breast cancer.[5] Althoughwell tolerated, tamoxifen is a partial agonist as well as antagonistof estrogen action. This can result in estrogenic effects on theuterus, leading occasionally to endometrial hyperplasia and can-cer.[6] In addition, tamoxifen may not be optimally effective incontrolling breast tumor growth because of its partial agonistaction. Inhibition of estrogen synthesis by selective inhibitors islikely to prevent the stimulatory effects of estrogen more com-

pletely,[1-4,7] as these compounds do not have agonist activity and

could therefore be more effective.To evaluate the antitumor efficacy of aromatase inhibitors,

tumors were grown from ER-positive human breast cancer cells

(MCF-7) in athymic (immunosuppressed) mice. Growth of these

tumors is dependent on stimulation by estrogen. Since estrogens

are produced from non-ovarian sources and are not under gonado-

tropin regulation in the postmenopausal patient, ovariectomized

mice were used. Very little peripherally formed estrogen is pro-

duced by these rodents. In order to provide a source of estrogen

to stimulate tumor growth, MCF-7 human breast cancer cells

stably transfected with the aromatase gene[8] (MCF-7Ca) were

used.[3,4] The parental MCF-7 cell line does not express

aromatase to any significant extent, as this gene tends to be un-

stable and may have been lost as a result of frequent passage in

culture. MCF-7Ca cells inoculated into each flank of the mouse

at four sites produce estrogens via aromatization. The cells are

responsive to the growth effects of estrogens via the ER. As

athymic mice secrete very low levels of adrenal androgens, they

are supplemented by administration of androstenedione. Tumors

form in 6–8 weeks. Although the model may produce a higher

level of estrogen than probably occurs in most patients, agents

studied in the model are likely to be more effective when antag-

onizing lower levels of estrogen. Since the tumors synthesize

estrogens and respond to their effects, this model allows us to

evaluate both aromatase inhibitors and antiestrogens and deter-

mine their optimal efficacy of use in sequence and/or combina-

tions. The objective of using this model is to provide information

that could be valuable as a basis for designing clinical trials. Since

clinical trials require a commitment of a large number of patients

as well as time and resources to provide statistically significant

results, preclinical data may serve to guide trial design and to

suggest whether a particular trial may be fruitful.

To date, recently produced clinical data have been consistent

with results obtained in this preclinical model. For instance, early

studies with the model showed that several aromatase inhibitors

were more effective than tamoxifen.[9,10] We also conducted pre-

clinical studies in the mouse xenograft model that are quite simi-

lar in design to the ATAC (Arimidex and Tamoxifen Alone or in

Combination) trial of anastrozole versus tamoxifen in the adju-

vant setting. As reported recently, the preliminary results of

ATAC have shown that a combination of tamoxifen with a non-

steroidal aromatase inhibitor was not significantly better than

using either of the drugs alone,[11] a result that was predicted by

the animal model a few years before.[12]

2. Studies with Aromatase Inhibitors

2.1 Comparison with Tamoxifen

In a study carried out over 9 weeks, letrozole was very effec-tive in inhibiting tumor growth, with maximum effect at 5 and 10μg/day in the mouse (figure 1). This effect is dose dependent inthe range of 1–10 μg/day/mouse and is maintained over thecourse of treatment. Animals (n = 4 or 5 per group) treated withletrozole 10 or 60 μg/day/mouse compared with tamoxifen 60μg/day/mouse experienced effective suppression of tumorgrowth compared with controls (figure 2).[10] In this early study,the two drugs, letrozole and tamoxifen, were effective in sup-pressing tumor growth compared with vehicle, but when tumorswere excised after 50 days of treatment, both letrozole doses (10and 60 μg/mouse/day) were significantly more effective thantamoxifen (60 μg/mouse/day) [p < 0.05] (figure 2).[10] In addi-tion, when half of the animals from among the control group weretreated with letrozole starting on day 30, by day 50 letrozole hadcaused tumor regression of these large tumors by about a third(figure 2).

One of the issues of concern with tamoxifen is its ability tostimulate endometrial tissue in some patients.[6,13] Therefore, inthe nude mouse xenograft, the effect of the agents on the endo-metrium was evaluated by measuring uterine weight. As expectedin these studies, the uterus responded to endogenously producedestrogens (control animals), whereas letrozole caused an inhibi-tory response that was not dose-dependent; both the 10 and 60μgdoses provided the same level of inhibition. However, tamoxifen

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Fig. 1. Letrozole dose response and antitumor effect. MCF-7Ca cells sus-pended in Matrigel were inoculated subcutaneously into ovariectomizedmice (1.75 × 106 cells per site, four sites per mouse). Beginning on thefollowing day, androstenedione (0.1 mg/mouse/day) was supplementedby subcutaneous injection. Treatment with letrozole (Let) 0.5–10μg/mouse/day was administered for 9 weeks (four mice per group).

2 Brodie et al.

© Adis Data Information BV 2003. All rights reserved. Am J Cancer 2003; 2 Suppl. 1

did not prevent estrogen action in the uterus, and tissue weightsin animals treated with this agent were comparable to thoseobtained in control animals (figure 3). The lack of antagonisticeffect of tamoxifen on the mouse uterus has been well docu-mented.[14]

We compared the effect of two aromatase inhibitors,anastrozole and letrozole (10 and 60 μg/mouse/day) with that ofthe pure ER blocker fulvestrant (five mice per group each withfour tumors). The mean tumor weight for letrozole (32.1 ±21.0mg), anastrozole (10 μg/mouse/day, 133.2 ± 22.0mg) andfulvestrant (114.9 ± 24.4mg) were significantly less than forthe controls (270.0 ± 27.7mg) [p < 0.05]. A dose response wasobserved with anastrozole (figure 4), but letrozole 10μg wasmore effective than the highest dose of anastrozole tested (60μg/day) after 4 weeks of treatment. The doses of anastrozole werenot significantly different, a result also observed in the clinic. Incontrast, a dose-response effect has been observed in patientstreated with letrozole.[15] In this mouse xenograft model, the pureantiestrogen fulvestrant was also effective, with an approximate20% reduction in tumor volume at 4 weeks. However, letrozolewas superior over that period of time, with approximately 70%decrease in tumor volume (figure 4).[10]

2.2 Combination Studies

Combining agents that inhibit estrogen biosynthesis withagents that cause estrogen-receptor blockade is a strategy thatlogically might be expected to lead to synergistic effects bytargeting these two independent mechanisms of action. In ashort-term experiment, we compared the effects of combininganastrozole with tamoxifen and also letrozole with tamoxifen. Asshown in figure 5, neither of these combinations was better than

the aromatase inhibitor alone.[12] This result was confirmed in theATAC trial of anastrozole in combination with tamoxifen.[11]

Subsequently, we evaluated letrozole alone or in combina-tion with tamoxifen over longer periods because of concerns thatshort-term experiments with low doses to investigate synergismmay not appropriately represent the clinical situation. Breast can-cer patients are treated with an agent until it is no longer effectiveand tumors progress. Therefore, in this experiment we set outto determine how long it would take for tumors to overcomethe growth inhibition imposed by each of these agents. Controltumors rapidly doubled in approximately 4 weeks, whereas alltreated groups showed a significantly longer tumor-doublingtime (figure 6, table I). Animals treated with tamoxifen experi-enced tumor-doubling times of approximately 16 weeks. How-ever, tumor-doubling time was still not reached with letrozole at35 weeks. Furthermore, the combined treatment of letrozole

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Fig. 2. Effects of letrozole and tamoxifen on the growth of MCF-7Ca tumors in nude mice. Groups of four mice were injected daily with letrozole (Let) 10 or60 μg/mouse/day or tamoxifen (Tam) 60 μg/mouse/day for 8 weeks. (a) Percentage change in total tumor volume over time according to treatment. (b) Meantumor weight ± SE at the end of the experiment.[10]

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Fig. 3. Comparison of effects of letrozole and tamoxifen on uterine weight.Uteri were removed from the same animals as in figure 2. **Uterine weights(mean ± SE) of letrozole-treated mice are significantly different from thecontrols (p < 0.01). [Adapted from Lu Q, Yue W, Wang J, et al. The effect ofaromatase inhibitors and antiestrogens in the nude mouse model. Breast CancerRes Treat, Vol. 50, 1998,[10] with permission from Kluwer Academic.]

Letrozole and Tamoxifen in a Xenograft Model for Breast Cancer 3

© Adis Data Information BV 2003. All rights reserved. Am J Cancer 2003; 2 Suppl. 1

10μg plus tamoxifen 60μg was only marginally better thantamoxifen alone, suggesting little benefit of the combination overtamoxifen, and it was clearly inferior to letrozole alone (tumor-doubling time 18 weeks versus not reached at 28 weeks; figure6, table I). Thus, the addition of tamoxifen to a nonsteroidalaromatase inhibitor did not improve the tumor growth inhibitionobserved with the aromatase inhibitor alone.[12,16-18]

2.3 Sequential Treatment

Patients with hormone-receptor-positive breast cancer areoften switched to secondary endocrine therapy when the initialtreatment fails to control tumor progression. For many years,

tamoxifen has been used as first-line endocrine therapy bothin the adjuvant and metastatic settings.[14] However, with the

demonstration of at least equivalence or superiority of the new-generation aromatase inhibitors to tamoxifen[19-23] and the ap-

proval of the former for first-line metastatic treatment, the issue

of treatment sequence with the available agents has arisen. The

possibility of delaying the development of resistance by alternat-

ing regimens of aromatase inhibitors and antiestrogens rather

than sequential use when resistance to one agent has developed

was investigated in the preclinical model.Using the murine tumor xenograft model, we could investi-

gate whether the overall outcome would be affected by the initi-

ating drug, that is, whether starting with an aromatase inhibitor

or with tamoxifen and alternating between the two therapieswould subsequently affect tumor response.

Animals were treated with either tamoxifen 100μg or letro-

zole 10μg or vehicle (control). In the control group, tumor size

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) Δ4A alone (100 μg/day)Letrozole (10 μg/day)Anastrozole (10 μg/day)Anastrozole (60 μg/day)Tamoxifen (60 μg/day) Fulvestrant (5 mg/week)

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Fig. 4. Effect of antiestrogens and aromatase inhibitors on breast tumorgrowth. Groups of four mice were injected subcutaneously with aromataseinhibitor or antiestrogen when tumor volumes reached approximately300mm3; all animals received androstenedione (Δ4A). Treatment was ad-ministered over a 4-week period. (Reproduced from Brodie et al.,[16] withpermission from Clinical Cancer Research.)

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Fig. 5. Effects of aromatase inhibitors combined with tamoxifen on tumorgrowth. Groups of five mice were injected subcutaneously with anastrozole,letrozole (5 μg/mouse/day) or tamoxifen (3 μg/mouse/day). The samedoses were used in combination. Treatment was administered over a 6-week period. (Reproduced from Lu et al.,[12] with permission.)

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Fig. 6. Effect of combining letrozole and tamoxifen on tumor-doubling time.Groups of 20 mice were injected subcutaneously with aromatase inhibitoror antiestrogen when tumor volumes reached approximately 300mm3. Ani-mals were treated with letrozole (Let) 10 μg/mouse/day and/or tamoxifen(Tam) 100 μg/mouse/day over a 28-week period. Δ4A = androstenedione.(Reproduced from Brodie et al.,[16] with permission from Clinical CancerResearch.)

Table I. Duration of effective treatment and corresponding tumor weightfrom mice treated with regimens of tamoxifen and letrozole

Treatment Tumor-doublingtime (w)

Mean tumor weights (mg)at 28w ± SE

Control <4 580.6 ± 150.0 (8w)

Tamoxifen 16 732.8 ± 303.3

Tamoxifen → letrozole 18 453.7 ± 139.4

Tamoxifen + letrozole 18 432.0 ± 119.2

Letrozole → tamoxifen 22 195.3 ± 39.1

Letrozole 35 Not determineda

a Animals were continued on treatment for additional weeks.

4 Brodie et al.

© Adis Data Information BV 2003. All rights reserved. Am J Cancer 2003; 2 Suppl. 1

doubled in less than 4 weeks. Other treatment groups includedtamoxifen initially, then switched to letrozole after 4 weeks andback to tamoxifen and so on every 4 weeks, and letrozole initiallythen alternating every 4 weeks with tamoxifen (figure 7). The

tamoxifen-alone and letrozole-alone groups were similar to thosedescribed above. In animals started on tamoxifen and alternatedbetween letrozole and tamoxifen every 4 weeks for the 28-weekstudy period, tumor growth oscillated between a decrease withletrozole and an increase with tamoxifen. Initiating treatmentwith letrozole resulted in longer control than initiating treatment

with tamoxifen (tumor-doubling time 22 vs 18 weeks; figure 7,table I). In addition, at 28 weeks, mean tumor weight was signif-icantly smaller for the group initially treated with letrozole (195.3± 39.1mg) compared with the group initially treated withtamoxifen (453.7 ± 139.4mg) [p < 0.05]. Nevertheless, none ofthese treatments was as effective as letrozole alone (table I). In

animals treated with letrozole alone, tumors had barely doubledin size at 35 weeks.[16,18] This suggests that switching from anaromatase inhibitor to tamoxifen or vice versa during long-termadjuvant therapy or upon progression in the metastatic settingmay lead to a worse outcome compared with achieving effectiveestrogen suppression with an aromatase inhibitor. This may resultin part from the partial agonist properties of tamoxifen. An indi-cation of this effect may have been observed in the recent cross-over phase III trial of tamoxifen versus letrozole in the first-linetreatment of metastatic breast cancer patients.[23]

These results have been confirmed in a second set of exper-iments where animals treated with tamoxifen were switched toeither letrozole or letrozole plus tamoxifen at 16 weeks (tumor-doubling time on tamoxifen). These animals achieved a goodsecondary response with letrozole, but a lesser response on thecombination of letrozole plus tamoxifen (figure 8). However, theresponse was less than observed with letrozole only administeredthroughout the same period.

3. Conclusions

The experiments described here were conducted in a murinexenograft model bearing human tumors that synthesize estrogensand are responsive to the effects of estrogen. In this system,letrozole was clearly more effective than tamoxifen with a muchlonger duration of response. This result has been confirmed clin-ically in the metastatic setting, although it should be borne inmind that this murine tumor model is more akin to the adjuvantsetting. The studies reported here suggest that early aromataseinhibitor treatment leads to a better and longer response.

Combination treatment does not seem to be any more effec-tive than a nonsteroidal aromatase inhibitor alone, as is evidentwith letrozole. Furthermore, alternating treatment between anantiestrogen and an aromatase inhibitor does not delay resistanceto treatment. The studies presented herein also show that in analternating paradigm, initial treatment with letrozole is signifi-cantly better than initial treatment with tamoxifen. This suggeststhat, although letrozole is very effective as second-line treatment,it is probably more appropriate to use as first-line treatment.

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Fig. 7. Effect of alternating letrozole and tamoxifen treatment every 4 weeks.The control, letrozole (Let) and tamoxifen (Tam) groups received daily sub-cutaneous injections over a 28-week period. The group initiated withletrozole was treated for 4 weeks and switched over to tamoxifen; treatmentwas then alternated between letrozole and tamoxifen every 4 weeks (thesingle asterisk indicates the switch to the alternative treatment). The sameprinciple was applied for the group initially treated with tamoxifen (20 miceper group). Δ4A = androstenedione. ** p < 0.05. (Reproduced from Brodieet al.,[16] with permission from Clinical Cancer Research.)

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Fig. 8. Effect of letrozole on tumors of tamoxifen-resistant mice. Mice wereinjected subcutaneously with tamoxifen (Tam) or vehicle when tumor vol-umes reached approximately 300mm3. At week 16, mice treated originallywith tamoxifen were switched to letrozole (Let) or tamoxifen plus letrozole(10 mice per group). (Reproduced from Brodie et al.,[16] with permission fromClinical Cancer Research.)

Letrozole and Tamoxifen in a Xenograft Model for Breast Cancer 5

© Adis Data Information BV 2003. All rights reserved. Am J Cancer 2003; 2 Suppl. 1

Acknowledgements

This work was supported in part by NIH CA62483 and by NovartisPharma, Basel, Switzerland. The authors have provided no information onconflicts of interest directly relevant to the content of this review.

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Correspondence and offprints: Dr Angela H. Brodie, Department of Pharma-cology and Experimental Therapeutics, School of Medicine, University ofMaryland, Baltimore, MD, 21201, USA .E-mail: abrodie@umaryland.edu

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© Adis Data Information BV 2003. All rights reserved. Am J Cancer 2003; 2 Suppl. 1