The Breast 18 (2009) 204-207
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`Contents lists available at ScienceDirect
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`The Breast
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`journal homepage: www.e|sevier.com/brst
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`*4
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`ELSEVIER
`
`Original article
`
`Bone turnover markers in postmenopausal breast cancer treated with
`fulvestrant — A pilot study
`
`A. Agrawala'*, R.A. Hannon b, I<.L. Cheung 3, R. Eastellb, J.F.R. Robertsona
`auniversity of Nottingham, Professorial Unit of Surgery, City Hospital Campus, Nottingham University Hospitals, Hucknall Road, Nottingham NC5 IPB, UK
`E University of Shefield, Academic Unit of Bone Metabolism, Metabolic Bone Centre, Sorby Wing, Northern General Hospital, Sheffield S5 7AU, UK
`
`ARTICLE INFO
`
`ABSTRACT
`
`Article history:
`Received 21 January 2009
`Received in revised form
`2 April 2009
`Accepted 16 April 2009
`
`Keywords:
`Bone markers
`Breast cancer
`L°Ca"Y advanced
`F“1"_e5”3“t
`A““e5”°g"-“
`
`Background: Tamoxifen has a protective effect on bone metabolism in breast cancer; aromatase inhib-
`itors deleterious and that offulvestrant is unknown.
`Methods: Fourteen locally advanced breast cancers with clinical benefit on fulvestrant (250 mg/month)
`as first—line primary endocrine therapy had sequential serum bone—specific alkaline phosphatase (BAP),
`N—terminal propeptide ofprocollagen type 1 (PINP) and C—terminal telopeptide (CTX) at 0,1, 5, 12, and 18
`months. Mean percentage changes (95% CI) were calculated.
`Results: Changes from baseline at 1, 5, 12, and 18 months with BAP (3.9—46.8 ng/ml) were +1.5 (+9.8 to
`+129), +2.2 (+22.1 to +266), +17.6 (+12.4 to +475), +1o.8 (+29.9 to +517); with PINP (20.6—82.1 ngl
`ml) were +3.4 (+12.0 to 19.0), +18.8 (+36.7 to +74.2), +475 (+21.4 to 115.3), +33.3 (+495 to +115.1)
`and with Cl'X (0.14+1.35 ng/ml) were +30.8 (0.1 to +61.6), +13.9 (+22.3 to +502), +429 (+127 to
`+98 5) +45 2 (+28 3 to +118 8)
`Conclusions: Long—term (18 months) stability of bone markers may be exploited by using fulvestrant
`earlier in sequence of endocrine therapies particularly in adjuvant setting in those with pre—existing
`cecreased bone mass.
`
`© 2009 Elsevier Ltd. All rights reserved.
`
`Background
`
`that accompanies declining
`increased bone turnover
`The
`estrogen levels at the onset of menopause in women leads to
`decreased bone mass and increased risk of fracture.
`In post-
`menopausal women with breast cancer this may be further aggra-
`vated by treatment with antiestrogen. Aromatase inhibitors such as
`anastrozole (Arimidexm, Astrazeneca), letrozole (Femaram, Novar—
`tis) or exemestane (Aromasinm, Pfizer) do not have any estrogenic
`agonistic activity and cause increased bone turnover resulting in
`significant loss in bone mass.1 Tamoxifen, however, affords some
`protection by virtue of its partial agonistic activity.2‘4
`
`Abbreviations: LAPC, locally advanced primary breast cancer; BAP, bone—specific
`alkaline phosphatase; PINP, N—terminal propeptide of procollagen type 1; CTX, C-
`terminal
`telopeptide; ER, estrogen receptor; T11’,
`time to progression; PgR,
`progesterone receptor; CB, clinical benefit; OR, objective response; MBC, metastatic
`breast cancer; SD, stable disease; CV, coefficient of variation; Gs, confidence
`intervals.
`* Corresponding author. Tel.: +44 115 82 31878/76; fax: +44 115 82 31877.
`E-mail addresses: amit.agrawal@nottingham.ac.uk (A. Agrawal), r.a.hannon@
`sheffield.ac.uk (R.A. I-Iannon), kl.cheung@nottingham.ac.uk (K.L Cheung), r.eastell@
`sheffield.ac.uk (R. Eastell), john.robertson@nottingham.ac.uk (_[.F.R. Robertson).
`
`0960-9776/$ ~ see front matter © 2009 Elsevier Ltd. All rights reserved.
`doi:10.1016/j.breast.2009.04.002
`
`Fulvestrant (Faslodexm, Astrazeneca) is a new estrogen receptor
`(ER) antagonist with no estrogen agonist effects5 and has a novel
`mode of action;
`it binds, blocks and increases degradation of ER
`protein, leading to an inhibition of estrogen signaling through the
`ER.6'7 In a prospectively planned combined analysis of the data from
`two randomized trials of similar design (Trials 20 and 21) fulvestrant
`was reported to be at least as effective as anastrozole in terms of time
`to progression (TIP; 5.5 months vs. 4.1 months, respectively).8 A
`subsequent prospectively planned, combined analysis of survival data
`reported that the median overall survival was not significantly
`different between the two treatments.9 In a further double—blind,
`randomized phase III trial (Trial 0025) fulvestrant (250 mg/month)
`was compared with tamoxifen (20 mg/day) in the first—line treatment
`of postmenopausal women with advanced breast cancer.1° Prospec-
`tive planned analysis of patients with ER and/or progesterone receptor
`(PgR) positive tumours ( ~ 80% of the population) showed median TTP
`of 8.2 months for fulvestrant and 8.3 months for tamoxifen with
`similar clinical benefit (CB) and objective response (OR) rates and
`overall survival between groups. However, to date there has been no
`data of the effect of fulvestrant on bone metabolism in humans.
`Bone is constantly renewed by the process of bone remodelling,
`in which old bone is resorbed by osteoclasts and replaced by new
`bone, which is laid down by osteoblasts. Markers of bone resorption
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`A. Agmwal et al. / The Breast 18 (2009) 204-207
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`205
`
`and formation, measured in serum or urine, reflect the activity of
`osteoclasts and osteoblasts, respectively. This study is the first to
`report the effect of fulvestrant on markers of bone turnover when
`used in postmenopausal women with locally advanced primary
`breast cancer (LAPC)
`in whom there was no evidence of overt
`metastatic disease.
`
`Materials and methods
`
`Patients
`
`Postmenopausal women with LAPC or metastatic breast cancer
`(MBC) received fulvestrant (250 mg) as their first—line primary
`endocrine therapy (so patients were endocrine naive) as part of an
`open—label prospective clinical trial that had received approval of
`the institutional Ethics Committee. Patients underwent staging
`investigations as per study protocol and included blood tests (full
`blood count, liver function tests, calcium, phosphate, CA15.3 and
`CEA), chest X—ray and pelvic X—ray for potential skeletal metastases.
`Bone scintigram was used if plain radiography was not definitive in
`diagnosing or
`ruling out metastases. Patients gave written
`informed consent for the trial including sequential serum samples
`and tissue biopsies. Twenty—five of 30 patients with LAPC/MBC who
`were recruited in this study had clinical benefit (CE). The remaining
`5 patients progressed within 6 months and were not included in
`the study. Of the 25 patients with CB, 2 males and 4 MBC patients
`were not
`included in the analysis. Thus, a series of 19 post-
`menopausal women with endocrine—na'1've LAPC (primary breast
`cancer> 5 cm and/or skin involvement) who had CB during ful-
`vestrant therapy were included. Patients with CB were selected so
`that any bone marker changes would reflect likely the activity of
`fulvestrant on bony tissue and not disease progression including
`bone metastasis (and so the MBC patients were excluded).
`Patients with LAPC had tumours of TNM stage Ilb, Illa or IIIb
`(Table 1). Fulvestrant (250 mg) was administered as a once—monthly
`intramuscular injection into the gluteus muscle. Patients had
`regular 3 monthly clinical examinations along with CA15.3 and CEA
`assessments. CB was defined as objective response (complete or
`partial response) or stable disease [SD] for 36 months‘ duration.“'12
`
`B0118 I’Tl(11’k€T'
`
`(ISSGSSITIGTIFS
`
`Sequential blood samples were taken at baseline and after 1, 6,
`12 and 18 months of fulvestrant treatment with majority of patients
`still being on treatment at 18 months. Patients were not strictly
`fasting though the large majority of samples were taken at the same
`time of the day (late mornings).
`The clotted blood samples were centrifuged (1000 g for 15 min),
`and the serum suitably aliquoted and stored at —2O “C. All samples
`taken from the same patient were analyzed in the same batch at the
`
`Table 1
`Patient and disease baseline characteristics.
`
`Median age, years (range)
`
`Tumour grade, n (%)
`1
`2
`3
`
`Estrogen receptor (ER) status
`Median ER H-score
`% Cells staining positive
`
`LAPC (n : 19)
`73.6 (549-909)
`
`4 (21.1)
`13 (68.4)
`2 (10.5)
`
`220
`100
`
`end of the study. Serum was analyzed for the following markers of
`bone formation and resorption.
`The bone formation markers, bone alkaline phosphatase (BAP)
`and N—terminal propeptide of procollagen type 1 (PINP), and the
`bone resorption markers were measured. Bone ALP, an isoenzyme
`of alkaline phosphatase, was measured using an automated
`chemiluminescent
`immunoenzymatic assay (Beckman Access
`OstaseTM 37300). Intra—assay coefficient of variation (CV) was <2.6%
`and the normal reference range for postmenopausal women was
`3.9—46.8 ng/ml. PINP, a by—product of type I collagen synthesis, was
`measured by a quantitative radioimmunoassay (Orion Diagnostica
`UniQTM PINP RIA). The intra—assay CV was 6.0% and the normal
`reference range for postmenopausal women was 20.6-82.1 ng/ml.
`Serum CTX, a degradation product of crosslinked type I collagen,
`was measured by an enzyme—linked immunoassay (Serum Cross-
`lapsTM, Nordic Bioscience Diagnostics). The intra—assay CV was 3.9%
`and the normal reference range for postmenopausal women was
`0.14-1.35 ng/ml.
`
`Statistical analysis
`
`Data were analyzed using Statgraphics PlusTM version 5 (Hern-
`don, VA)
`statistical
`software. Data are presented as mean
`percentage change (from baseline)
`in marker level with 95%
`confidence intervals (Cls).
`
`Results
`
`The patient and disease characteristics are shown in Table 1. The
`median duration of CB for patients receiving fulvestrant was 280+
`months (range: 10.9—55.4 months;
`treatment ongoing in 15
`patients at 18 months and in 11 patients at the time of analysis).
`There were no ‘baseline’ data for 5 patients in whom a sample of
`blood at baseline was not available. Therefore, 14 patients had bone
`marker measurements at baseline, 1, 6, 12 and 18 months. Mean
`percentage change (from baseline) in serum PINP, bone ALP and
`CTX levels in these 14 patients is shown in Table 2. Wilcoxon signed
`rank test did not show any significant difference from baseline at
`any time—point for any of the 3 markers in these patients.
`Of the 5 patients who did not have baseline sample available,
`the marker assessment was over a 17-month period from 1 to 18
`months. l(rusl<al—Wallis analysis revealed no significant changes in
`bone markers between any of the time—points over this 17-month
`period in these patients. Similarly, in all 19 patients with LAPC, no
`significant changes were apparent over the 18-month period.
`
`Discussion
`
`LAPC patients who had shown CB were selected for this study so
`that bone turnover marker levels being estimated were not
`confounded by the presence of overt or occult progressive bony
`metastases. Furthermore since median time to progression of
`disease was about 24 months, only samples collected in the first 18
`months of the trial were used for marker assessments. This was to
`avoid as far as possible confounding the results with any early
`biochemical evidence due to undiagnosed progression of occult
`bony metastases or the development of new overt bony metastases.
`The chosenbone formation and resorption markers are established
`markers of bone turnover which have been validated in several
`
`studies.” Although bone markers have high intra—individual variability
`and diurnal variation (especially CTX)” they provide more dynamic
`and earlier measurement of the skeletal status when compared with
`bone mineral density measurement.15"6 Serum markers, however,
`exhibit less intra—individual variation than urinary markers.”
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`A. Agrawal et ul. / The Breast 18 (2009) 204-207
`
`Table 2
`Mean and CI (95% confidence interval) for LAPC (n : 14) patients.
`Marker: pre—treatrnent (n : 14)
`1 Month (n : 13)
`Bone ALP
`+1.5 (+9.8 to +12.9)
`PINP
`+3.4 (—12.0 to 19.0)
`Cl‘X
`+30.8 (0.1 to +616)
`
`6 Months (11 :14)
`+2.2 (—22.1 to +266)
`+18.8 (—36.7 to +742)
`+139 (+223 to +502)
`
`12 Months (n :11)
`+17.6 (—12.4 to +47.6)
`+47.5 (—21.4 to +116.3)
`+ 42.9 (—12.7 to +985)
`
`18 Months (n :10)
`+10.8 (—29.9 to +51.7)
`+33.3 (—49.5 to 116.1)
`+45.2 (—28.3 to +118.8)
`
`In these LAPC patients with no demonstrable bony metastases,
`the stability of bone turnover markers over 17-18 months period
`(Table 2) suggests the apparent lack of effect of fulvestrant on bone
`turnover. This was further supported by data in the further 5
`patients with unavailable baseline serum sample, in whom there
`was no significant difference between any ofthe time—points over 17
`months period. To the best of our knowledge, there is no known data
`in literature of long—term effect of fulvestrant on bone turnover in
`human studies. Reports of its effect in animals do exist but the data
`are conflicting. In an experiment by Gallagher et al.17 in adult female
`intact
`rats,
`fulvestrant
`reduced cancellous bone volume by
`increasing bone resorption and decreasing bone formation by
`abolishing protective effect of estrogen. The increase in bone
`formation indices was not seen in ovariectomised rats. However, it
`did not affect longitudinal or periosteal tibial growth in either
`ovary—intact or ovariectomised rats given estradiol or vehicle.
`Sibonga et al.18 in a study of cancellous bones in adult rats found that
`fulvestrant increased skeletal indices of bone turnover in ovary-
`intact rats with a reduction in cancellous bone area. However, in
`ovariectomised rats there was a reduction in bone turnover that was
`associated with an increase in bone area. Thus if the data from the
`above experiments in ovariectomised rats are extrapolated to
`postmenopausal women as in our study then there is no clear
`evidence for negative influence of fulvestrant on bone tissue.
`In yet another study in rats, Lea et al.19 administered fulvestrant
`alone and in combination with the anti—androgen, bicalutamide
`(CasodexTM, AstraZeneca, US) and compared the effects on the
`skeleton with those of ovariectomy. They reported that ovariec-
`tomised rats lost significantly greater cancellous bone volume
`compared with those treated with fulvestrant alone. The combi—
`nation of fulvestrant and bicalutamide, however, resulted in bone
`loss equivalent
`to that
`in ovariectomised animals. The study
`authors concluded that ovarian androgens possibly protect against
`bone loss in rats made estrogen deficient otherwise by fulvestrant.
`This again if extrapolated to our postmenopausal women may
`mean that even if there was bone loss induced by fulvestrant by
`virtue of it being a pure antiestrogen with no agonistic activity
`(unlike tamoxifen), ovarian androgens may alone have protected
`against significant bone loss. In contrast to fulvestrant, possible
`protective effect of androgens on bone is lost on treatment with
`aromatase inhibitors due to blockage of conversion of circulating
`androgens into estrogens (by aromatase inhibitors).
`In a multi—centre randomized study by Donnez et al.,2° 50 pre-
`menopausal women had short—term exposure to 3 doses of fulves-
`trant (50 mg, 125 mg, or 250 mg) as an intramuscular injection over
`12 weeks period and compared with goserelin and placebo in
`reduction of uterine fibroid growth before planned hysterectomy.
`The primary safety end—point of bone resorption measured by
`urinary crosslinked N—telopeptide and free deoxypyridinolone were
`measured at baseline, 5, 9 and at 13 weeks (completion of study).
`There was little change in median bone resorption indices from
`baseline and in fact no statistical difference between various doses
`of fulvestrant and placebo. A recent phase II neoadjuvant trial
`(NEWEST) in 211 postmenopausal women with ER positive large
`primary breast cancers randomised patients into those receiving
`approved dose of fulvestrant (250 mg) versus loading dose (500 mg
`including additional 500 mg on day 14 of first month) over a period
`of 16 weeks. This trial compared serum bone markers (BAP,
`
`PINP, CTX) besides the main tissue tumour indices. The study
`investigators reported no change in bone markers with either
`dose.” This recent presentation of the NEWEST results at the San
`Antonio Breast Cancer Conference supports the findings of this
`study. However, our study remains the only long—term data on the
`effect of fulvestrant on markers of bone metabolism.
`Journe et al.22 showed that ibandronate (a bisphosphonate)
`enhanced the growth inhibitory action of tamoxifen and fulvestrant
`in estrogen—sensitive MCF—7 breast cancer cells. The combination
`analysis identified additive interactions between ibandronate and ER
`antagonists. However, in the clinical setting it remains to be seen
`whether or not
`there is additive efficacy of fulvestrant plus
`a bisphosphonate in the treatment of bony metastases.” On the
`other hand the apparently neutral effect of fulvestrant on bone
`metabolism makes either a higher dose of fulvestrant alone or ful-
`vestrant plus anastrozole combination, a potentially attractive
`option for future adjuvant endocrine therapy.
`
`Conclusions
`
`In this small patient series and within the limitations of inter-
`preting variability of response of bone markers, there was a lack of
`change in markers equating to long—term stability of bone turnover
`markers in postmenopausal women with LAPC treated with ful-
`vestrant for over a period of 18 months. This is in contrast to the
`increase in bone markers (serum BAP, PINP and CTX) at 12 months
`compared to the baseline seen in 58 patients who received anas—
`trozole in a sub—protocol study of patients in ATAC trial.1
`Data from both animal and now human experiments portray
`a favourable profile of fulvestrant on bone tissue. While this is the
`first published report of the effects of fulvestrant on bone metab-
`olism in humans,
`the recent San Antonio presentation has
`confirmed that fulvestrant appears neutral
`in respect of bone
`metabolism. Furthermore the present study is the only one which
`has assessed the long—term effects of fulvestrant on bone metabo-
`lism. The possible lack of effect on bone turnover may be exploited
`clinically in the future especially in the adjuvant setting. However,
`larger randomized studies including head—to—head comparison of
`long—term bone turnover effects of fulvestrant with tamoxifen and
`aromatase inhibitors are required to confirm these findings. The
`comparison could be more robust with inclusion of bone mineral
`density measurements along with serum samples as radiographs
`and tumour markers alone may not be sensitive enough.
`
`Conflict of interest statement
`
`Prof. Robertson and Dr. Cheung have received honoraria and
`funding from AstraZeneca. Prof. Eastell is a consultant to Astrazeneca.
`Prof. Eastell and Dr. Hannon have received honoraria and funding
`from AstraZeneca. Dr. Agrawal has been sponsored by AstraZeneca
`for Scientific Meetings in the past.
`
`Authors’ contributions
`
`Laboratory tests were arranged and provided by Dr. RA Hannon
`and Prof. R Eastell. Dr. RA Hannon provided statistical help.
`Dr. A Agrawal drafted the manuscript which has been revised and
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`A. Agrawal et al. / The Breast 18 (2009) 204-207
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`207
`
`approved by Prof. JFR Robertson, Dr. RA Hannon, Prof. R Eastell
`and Dr. KL Cheung.
`
`Funding source
`
`The original clinical trial was funded by Astrazeneca, UK. This
`subset study (including laboratory tests for the bone markers),
`however, was funded by the Division of Breast Surgery, University
`of Nottingham, United Kingdom.
`
`Ethical approval
`
`The original clinical trial and subset studies were approved by
`the Local Research Ethics Committee, Nottingham, UK.
`
`Acknowledgements
`
`their gratitude to Nicola Linley
`The authors express
`(University of Nottingham, UK) for the preparation of serum
`samples and Fatima Gossiel (University of Sheffield, UK) and
`Julie Porter (University of Sheffield, UK) for running the bone
`marker assays.
`
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