Biological activity of anastrozole in postmenopausal patients with advanced breast cancer: effects on estrogens and bone metabolism

E. Bajetta1,+, A. Martinetti2, N. Zilembo1, P. Pozzi1, I. La Torre1, L. Ferrari2, E. Seregni2, R. Longarini1, G. Salvucci1 and E. Bombardieri2

1 Medical Oncology Unit B and 2 Nuclear Medicine Unit, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy

Received 12 July 2001; revised 17 September 2001; accepted 2 October 2001


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Background:

To study the short-term biological effect of anastrozole on serum estrogens, androgens, 17-hydroxyprogesterone (17OH-PGR), gonadotrophins, sex hormone binding globulin (SHBG) and bone metabolism markers.

Materials and methods:

Thirty-four consecutive patients with advanced breast cancer received anastrozole 1 mg/day. Blood samples were taken before commencement of treatment and at 2, 4, 8 and 12 weeks during treatment to measure serum levels of estrogens (E1, E2 and E1-S), androgens [androstenedione ({Delta}4), dihydrotestosterone (DHT), testosterone (TST), free TST, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S)], 17OH-PGR, SHBG and gonadotrophins. As an indicator of bone resorption, we measured serum levels of C-terminal telopeptide of type I collagen (ICTP) and the cross-linked N-telopeptide of type I collagen (NTx), and for osteoblastic activity, intact osteocalcin (BGP) and bone alkaline phosphatase (BAP).

Results:

After 2 weeks E1 and E1-S levels decreased on average by 56% (range 23.1–88.8%) and 75.8% (range 52.4–87.2%), respectively; E2 decreased on average by 62% (range 31.4–89.6%). No significant changes were detected in levels of androgens or 17OH-PGR. There was a significant increase in gonadotrophins over time (P = 0.0001 for both luteinizing hormone and follicle-stimulating hormone), and a significant decrease in SHBG (P = 0.0001). A progressive significant increase in bone metabolism serum markers was detected in all patients: BAP, P = 0.039; BGP, P = 0.016; ICTP, P = 0.0021; and NTx, P = 0.0013. In particular, patients with bone metastases had a statistically significant increase of bone resorption markers (ICTP, P = 0.0019; NTx, P = 0.025) and borderline for bone formation markers. In patients without bone disease, BAP, BGP and ICTP remained unchanged, whereas serum NTx significantly increased (P = 0.019).

Conclusions:

Anastrozole is a selective aromatase inhibitor as it does not modify serum levels of androgens and 17OH-PGR. In our experience no relationship was found in the short-term period between serum estrogen suppression and bone metabolism.

Key words: androgens, aromatase inhibitors, bone metabolism markers, breast cancer, estrogens


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Aromatase inhibitors are currently used for treating postmenopausal advanced breast cancer (ABC) patients suitable for hormone therapy. These drugs have shown superiority over progestins [14] in a second-line therapeutic setting.

Anastrozole is a non-steroidal aromatase inhibitor that has recently been shown to be superior to tamoxifen when given as first-line treatment [57]. The pharmacological profile of anastrozole has been described previously [8]. Anastrozole can suppress circulating estrogen levels to a similar extent as letrozole, vorozole and exemestane, but to a greater extent than formestane [9]. This suppression is considered to be the major pharmacological activity of aromatase inhibitors and is responsible for their clinical efficacy in estrogen-dependent tumours, although no correlation has been demonstrated between estrogen suppression and clinical response [10]. However, in breast cancer patients, the biological activity of aromatase inhibitors is highly complex—these drugs have been shown to affect other hormones and growth factors—although the clinical significance of this has not yet been elucidated. Furthermore, it has been demonstrated [11, 12] that steroid hormones in women play a key role in regulating the equilibrium between bone formation and bone resorption, and the reduction in circulating estrogen levels induces frailness or loss of bone, as physiologically demonstrated in postmenopausal healthy women [6, 8].

The aim of this study was to assess the effects of anastrozole, when used in postmenopausal ABC patients on levels of serum estrogens, androgens, luteinizing hormone (LH), follicle-stimulating hormone (FSH), sex hormone binding globulin (SHBG) and 17-hydroxyprogesterone (17OH-PGR). In addition, the changes in bone formation and resorption, as possible consequences of estrogen suppression induced by anastrozole, have been studied. Thus, using bone formation markers, we have evaluated the circulating levels of osteocalcin (BGP) and the bone-specific isoform of alkaline phosphatase (BAP). Using bone resorption markers, we have evaluated the circulating levels of the C-terminal telopeptide of type I collagen (ICPT) and the cross-linked N-telopeptide of type I collagen (NTx). These markers were evaluated in all patients, irrespective of the presence of bone metastases.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
From January 1998 to May 1999, 34 consecutive postmenopausal patients with histopathologically assessed ABC entered into the trial. All patients received anastrozole (Arimidex®; AstraZeneca, Basiglio Milano, Italy) 1 mg/day (p.o.). Postmenopausal status was defined by one of the following criteria: (i) no spontaneous menses for at least 5 years; (ii) spontaneous amenorrhoea lasting at least 12 months with gonadotrophin levels within the postmenopausal range (20–143 mIU/ml FSH and 16–71 mIU/ml LH, respectively); and (iii) secondary amenorrhoea due to surgical treatment or radiotherapy.

Patients were required to be estrogen receptor and/or progesterone receptor positive. Patients with unknown receptor status were included in the trial if they had achieved an objective response after a previous hormonal treatment, with a disease-free interval of >=2 years. Previous chemotherapy and/or hormonal therapy for advanced/metastatic disease were permitted. The use of biphosphonates was not allowed. Before entering the study, the patients had to be in progression of disease and any previous treatment had to have been withdrawn more than 3 weeks earlier. In addition, patients had to have either one bi-dimensionally measurable or one evaluable bone lesion, and a performance status <=2 according to the World Health Organization (WHO) score.

Patients were excluded if they had received prior treatment with any aromatase inhibitor, and concomitant treatment with corticosteroids was not allowed. Patients were also ineligible if they suffered from endocrine disorders and/or impaired liver or kidney functions. Staging and tumour response were assessed by means of physical examination, bone scan, chest and skeletal radiographs, liver echography or computed tomography scan, whole blood cell count and blood chemistry. These examinations were performed at the beginning of the study and every 12 weeks thereafter.

Signs, symptoms and toxicity were evaluated according to the National Institute Criteria, and clinical response was evaluated in accordance with the UICC (Union Internationale Contre le Cancer) criteria. Because hormone-based therapy has mainly a cytostatic effect, we also considered stable disease lasting >24 weeks.

Since this paper deals with the biological effects of anastrozole in the short term, the clinical response considered was that obtained at 24 weeks from the beginning of the study. All patients gave their written informed consent before study enrolment; the trial was approved by the local BioEthical Committee and conducted according to good clinical practice requirements.

Biological samples, timing and specimen collection
Peripheral blood samples were collected at baseline and at 2, 4, 8 and 12 weeks from therapy initiation between 9.00 a.m. and 10.00 a.m., before drug intake and after a mandatory 12-h fasting period. Serum separation was achieved by centrifugation of the blood sample (room temperature, 1500 g for 15 min); finally, the supernatants were divided into aliquots and stored at –60°C until the day of the assay. All hormone assays were performed by the Laboratory of Nuclear Medicine Unit (Istituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy).

All assay kits were assembled according to the manufacturer’s guidelines. Each assay was validated using a recovery test by spiking and dilution. The accuracy of these assays was tested against serum samples using known concentrations of the tested analytes. The assays were performed in duplicate and all samples from the same patients were analysed in the same assay batch. The assays had intra-assay coefficients of variation that were <5% and interassay coefficients of variation <6%.

Hormonal measurement
Serum gonadotrophins (LH and FSH) were measured by immunoenzymatic assay (Abbott, Roma, Italy). Serum dihydrotestosterone (DHT) was measured by immunoenzymatic assay (DRG Instruments, Torino, Italy). Serum testosterone (TST), free TST, androstenedione ({Delta}4), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), SHBG and 17OH-PGR levels were measured by radioimmunometric assay (RIA) DPC (Diagnostic Products Corporation; Medical Systems, Genova, Italy). Estrogen serum levels (estrone E1, estradiol E2 and estrone sulfate E1-S) were measured using RIA after chromatographic separation, as previously reported [13].

Bone metabolism evaluation
As an indicator of bone formation activity, we measured BGP and BAP by immunoenzymatic assay (Biosystem, Milano, Italy). As an indicator of bone resorption, we evaluated serum levels of ICTP and cross-linked NTx. ICTP was measured by RIA (Orion) purchased from Metra Biosystem (Milano, Italy). NTx was measured by immunoenzymatic assay (Ostex International) purchased from Bouty (Milano, Italy).

Statistics
This was a prospective, non-comparative trial, and only a descriptive analysis was performed. Tumour response is reported as rate; quantitative data are reported as median (range). The U-paired test was used to compare the values of each analyte at baseline with those after 12 weeks. Correlation analysis among continuous variables was performed quantifying the strength of the associations by calculating the Pearson’s correlation coefficient (r) and the non-parametric Spearman’s rank correlation coefficient (rho). In all statistical tests performed, a 5% level of significance was used. The software SPSS 6.0 for Windows (SPSS, Chicago, IL) was used for data management and to perform statistical calculations.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Thirty-four patients were enrolled and suitable for evaluation; their characteristics are shown in Table 1. We have reported the results obtained from all patients, and also separately for patients with (group A, 22 patients) and without bone metastases (group B, 12 patients).


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Table 1.  Patient characteristics
 
Biological assessment
Table 2 summarises the results of all the biological variables evaluated during the study.


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Table 2. Serum levels of the evaluated analytes as median values (range)
 
Estrogen levels
Anastrozole treatment significantly (P = 0.0001) suppressed serum E1, E2 and E1-S levels, and this activity was evident after just 2 weeks. E1 levels decreased on average by 56.5% (range 23.1–88.8%), E2 on average by 62.6% (range 31.4–89.6%) and E1-S levels by 75.8% (range 52.4–87.2%).

One patient, who had high serum estrogen levels at baseline, experienced variable suppression during treatment with high values after 8 weeks, and only after 12 weeks were the estrogen levels lower than baseline values. No correlation was observed between estrogen suppression and clinical response. No statistically significant differences in baseline serum E1, E2, E1-S levels were observed for patients of groups A and B, and the estrogen suppression was similar in each group. Figure 1 shows the degree of estrogen suppression in patients of groups A and B observed during the study.



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Figure 1. Median serum levels (relative change versus baseline) of (A) E1, (B) E2 and (C) E1-S for patients with (group A) and without (group B) bone metastases.

 
Gonadotrophin levels
There was an increase in serum LH and FSH levels that was statistically significant (P = 0.0001 for each group) after 4 weeks of treatment.

Androgen levels
Androgens did not show any significant change during treatment and no correlation was found with tumour response. Similar results were observed in patients of groups A and B. Furthermore, 17OH-PGR serum levels did not significantly change after 12 weeks. One patient, who had high serum estrogen levels at baseline, had a similar level of androgen. There was a progressive decrease in serum SHBG levels, which became statistically significant after 12 weeks (P = 0.0001).

Bone marker levels
We observed a statistically significant increase in bone formation markers (BAP, P = 0.039; BGP, P = 0.016) and bone resorption markers (ICTP, P = 0.0021; NTx, P = 0.0013) after 12 weeks when all patients were analysed in a single group. Table 3 shows the levels of all bone metabolism markers in patients from groups A and B. There was no difference between the two groups regarding serum BGP and ICTP levels, either at baseline or during treatment, although there was a difference in baseline values for BAP (P = 0.022) and NTx (P = 0.011), which significantly increased during treatment (P = 0.009 and 0.007, respectively).


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Table 3.  Median levels (range) of serum bone metabolism markers in 22 patients with bone metastases (group A) and in 12 patients without bone metastases (group B)
 
In group A, the serum levels of all bone metabolism markers increased during treatment. This increase was statistically significant for bone resorption markers (ICTP, P = 0.0019; NTx, P = 0.025) and borderline for bone formation markers (BAP, P = 0.067; BGP, P = 0.053).

In group B, the levels of bone metabolism markers remained unchanged for BAP, BGP and ICTP, although the increase in NTx serum levels was statistically significant (P = 0.019). It is worth noting that in this group, two patients with only soft tissue metastases had serum NTx levels of 13.47 and 22.85 nMBCE, respectively, at baseline. After 24 weeks, the latter patient had NTx levels up to 31.4 nMBCE and the onset of bone lesions; the other patient had 23.1 nMBCE and progressed in bone 3 months thereafter.

Figure 2 shows the median value of the bone metabolism markers in the patients of groups A and B and according to the objective response.



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Figure 2. Median serum levels of (A) bone alkaline phosphatase, (B) osteocalcin, (C) C-terminal telopeptide of type I collagen (ICTP) and (D) cross-linked N-telopeptide of type I collagen (NTx) for patients with bone metastases relative to the objective, and for patients without bone metastases.

 
Correlation analyses
As expected, estrogen and androgen levels appear to be correlated with each other at baseline (data not shown).

Table 4 shows the correlations between estrogens and bone markers at baseline in all patients. No correlations were ob-served between estrogens and all bone metabolism markers; however, significant correlations were found between markers of bone formation (BGP and BAP, r = 0.36, P = 0.033) and markers of bone resorption (ICTP and NTx, r = 0.42, P = 0.013). Furthermore, BAP and markers of bone resorption (ICTP and NTx) are positively correlated (r = 0.37, P = 0.027 and r = 0.58, P = 0.0001, respectively).


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Table 4.  Spearman’s correlation coefficients (rho) and the corresponding P value, among baseline circulating levels of estrogens and bone metabolism markers in all patients
 
At baseline, in patients from group A, we found a correlation between markers of bone formation (BGP and BAP, P = 0.04), and a statistically significant correlation between BAP and NTx (P = 0.0001). In contrast, during treatment, the correlation between markers of bone formation disappears but there was a statistically significant correlation between markers of bone resorption (ICTP and NTx, P = 0.0001). A good correlation was also present between BAP and markers of bone resorption (ICTP and NTx, P = 0.008 and 0.001, respectively). No correlation was found between bone metabolism markers either at baseline or during treatment in group B patients.

Tumour response
After 24 weeks of treatment, two patients had a complete response (5.9%), six patients a partial response (17.6%) and 15 patients a stabilisation of disease (44.1%). The overall response rate was 23.5% and the clinical benefit rate was 67.6%.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The use of new generation aromatase inhibitors in the treatment of postmenopausal ABC patients with estrogen-dependent tumours is on the increase [14]. Among those aromatase inhibitors available, anastrozole has a well-established role in clinical practice for both advanced disease and first-line treatment.

The major pharmacological activity of anastrozole is the suppression of serum estrogen levels; however, few data are currently available on the other biological effects of this drug. In this paper, we have analysed the biological profile of anastrozole by evaluating serum estrogen, androgen, gonadotrophin and SHBG levels in ABC patients. Furthermore, bone metabolism markers have also been evaluated as there is a relationship between estrogen suppression and bone loss.

The results of this study confirm the efficacy and selectivity of anastrozole in estrogen suppression, as no changes in serum androgens or 17OH-PGR levels were detected, despite significant effects on serum E2, E1 and E1-S levels. The degree of estrogen suppression was similar to that reported with exemestane [15, 16], confirming a similar potency of steroidal and non-steroidal compounds. It is widely accepted that estrogen supply is necessary for tumour-cell growth. However, the relationship between the degree of estrogen suppression and tumour response is still unclear [10], as also confirmed by the results of this study, which do not show any correlation between estrogen suppression and objective response.

A statistically significant increase in serum gonadotrophin levels was observed during treatment, a finding that has already been reported in other studies with aromatase inhibitors [17]. This increase is likely to be due to the removal of estrogen negative feedback mechanisms, which normally serve to inhibit gonadotrophin secretion.

Regarding serum SHBG levels, it is known that these reflect the estrogen/androgen balance, as its synthesis is stimulated by estrogens and inhibited by androgens. It is likely that the marked estrogen suppression induced by anastrozole is the mechanism responsible for the decrease in serum SHBG levels in our patients. We have observed a similar outcome in two previous studies carried out with other aromatase inhibitors [16, 18].

The most interesting result of this study was the behaviour of bone metabolism markers. Normally, bone tissue is continuously remodelled by resorption and formation. This physiological balance is generally referred to as coupling, and guarantees the maintenance of a stable bone mass. In women, steroid hormones play a key role in regulating the equilibrium between osteoblastic and osteoclastic activity, as demonstrated by the observation that the suppression of circulating estrogens induces bone loss [6, 8]. In particular, in ABC patients, the occurrence of bone metastases unbalances this equilibrium and leads to uncoupling of the metabolic process, with a resorptive (osteolytic) or formative (osteoblastic) component, depending on the type of metastases. Metastatic bone destruction is initiated by the activation of local osteoclasts, which are themselves induced by various locally and systemically acting oncogenic mediators. Assessment of bone turnover using a panel of specific markers can be helpful for the detection of bone turnover changes in the elderly, for monitoring the effect of therapy and for the study of bone metastases in patients affected by various neoplasms.

Here we have evaluated two markers of bone resorption activity [ICTP and NTx (as a marker of matrix degradation] and two markers of bone formation activity [BAP (as a marker of organic bone matrix synthesis) and BGP (as a marker of the bone mineralisation process)]. We have evaluated the behaviour of bone metabolism markers in patients with bone metastases as markers of clinical evolution, and in patients without bone metastases to monitor the effect of therapy. In our series of patients we found no relationship between bone markers and aromatase inhibition. However, we found that BAP and NTx were correlated with the presence of bone metastases and their clinical evolution, suggesting their usefulness in the monitoring of disease outcome. Furthermore, the behaviour of NTx in the two patients of group B that developed bone metastases thereafter suggests that it might be useful for predicting the onset of bone lesion in patients without skeletal disease. We have observed the elevation of all bone marker serum concentrations in patients with progressive disease, and this finding confirms the fact that either bone resorption or bone formation are increased during the development of skeletal metastases, although the statistical analysis could be biased by the small number of patients (Figure 2). This can be demonstrated by the presence of mixed (osteolytic and osteoblastic) metastases, or by the attempt to maintain the coupling of bone formation and resorption [1921].

It is worth noting that high baseline ICTP values were predictive of poor prognosis. Similar findings were reported by other authors for other neoplasms. In this respect, Elomaa et al. [22] reported that ICTP concentration was a prognostic indicator for multiple myeloma and Kylmälä et al. [23] obtained similar results for prostate cancer. The prognostic significance of ICTP could reflect a more aggressive behaviour that could be sustained by tumour cells with a high proliferation rate, or by an enhanced activation of osteoclasts induced by the tumour itself. Serum BAP levels displayed similar behaviour, and the pretreatment value of BAP seemed to be related to survival, as described in other studies [24, 25].

In conclusion, the results of this study clearly demonstrate for the effectiveness and selectivity of anastrozole in suppressing estrogen levels, and show that, for the limited time period of our analysis, there was no correlation between bone markers and aromatase inhibition.


    Acknowledgements
 
The authors would like to thank the Italian Trials in Medical Oncology (ITMO) Scientific Service for their editorial assistance and Dr R. Artioli (AstraZeneca) who partially supported this research.


    Footnotes
 
+ Correspondence to: Dr E. Bajetta, Medical Oncology Unit B, Istituto Nazionale per lo Studio e la Cura dei Tumori of Milan, via G. Venezian 1, 20133 Milan, Italy. Tel: +39-02-23902500; Fax: +39-02-23902149; E-mail: bajetta@istitutotumori.mi.it Back


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
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