C. Recchione,1 E. Venturelli,1 A. Manzari,2 A. Cavalieri,1 A. Martinetti1 and G. Secreto1*
1Endocrine Unit and 2Division of Atedical Statistics and Biometry, National Cancer Institute, 20133 Milan, Italy
Correspondence-G. Secreto.;
Abstract
The ability of breast tumours to synthesize hormones is well recognized, and local production of sex steroids is thought to play a role in breast cancer growth. We measured the intratumour and circulating levels of testosterone, dihydrotestosterone (DHT) and oestradiol in 35 histologically confirmed carcinomatous mammary tissues obtained at breast surgery from 34 postmenopausal patients, age 50-85 years. Intra-tissue steroids were extracted with ethanol:acetone (1:1; v/v), defatted with 70% methanol in water, and extracted with ether. Steroids, from tissue and serum, were separated by partition chromatography on celite columns and were measured by RIA. Intratumour testosterone and DHT concentrations were significantly correlated, after the exclusion of an outlier (rs = 0.71; P — 0.0001). No association was found between oestradiol and either of the two androgens. Mean oestradiol and DHT concentrations were significantly higher in tissue than in blood (P = 0.0001). Mean testosterone levels in tissues did not significantly differ from those measured in blood. Our data suggest that at least a part of intratissue DHT is produced locally from testosterone. The meaning of high oestradiol and DHT levels in cancer tissue still needs to be defined.
Introduction
The ability of breast tumours to synthesize hormones is well recognized, and it is assumed that the total amount of a steroid in breast cancer tissue is provided in part by uptake from plasma and in part by local production. Breast cancer growth is influenced by sex hormones, and hormone levels in cancer tissues are considered to reflect their biological activity better than their levels in blood.
The intratumour concentration of oestrogens has been extensively studied, and particular interest has been addressed to the measurement of adrenal androgens [dehydroepiandrosterone-(sulphate), androstene-diol and androstenedione], which can serve as substrates for aromatization to oestrogen [1-8]. Although the presence of androgen receptors in breast tumours [9] indicates a possible role for androgens in regulating cancer growth, only a few papers have dealt with testosterone levels in breast cancer tissues [3, 10], and measurement of dihydrotestosterone (DHT) concentrations has been reported only by Mistry et al. [10]. A wide range of testosterone values was found in breast cancer tissue, with a tissue/plasma ratio of 0.05-5.0 [3]. At target tissues, testosterone can act as a full hormone or can serve as a pro-hormone for aromatization to oestradiol by the enzyme aromatase or conversion to DHT by the enzyme 5α-reductase. Aromatase and 5α-reductase activities are present in mammary tumours, and a role of testosterone in the intratumour synthesis of one or the other metabolite can be tentatively inferred. However, whereas testosterone is the obliged precursor of DHT, oestradiol can be produced alternatively by reduction of the ketonic group of oestrone.
In the present report, we measured the intratu-moural and the circulating levels of testosterone, DHT and oestradiol in postmenopausal breast cancer patients. The aim of the study was to support and add to previous findings in the literature [3, 10].
Experimental
Patients and sample preparation and storage
Carcinomatous mammary tissue was obtained at breast surgery from 34 consecutive patients bearing tumours larger than 3 cm, without clinical or radio-graphic evidence of distant métastasés. Two primary carcinomas were found in the same breast of one patient, and they were processed separately as 2 different tumours. All patients were postmenopausal, and their ages ranged between 50 and 85 y (mean + SD, 64.4 + 8.2). None had received any hormonal preparation for the last 6 months, and none had been previously treated with chemotherapy or hormone therapy for their disease.
Immediately after removal, a portion of the tumour was cut off, accurately dissected free of fat, pulverized with a micro-dismembrator and stored at — 80°C until analysis. The diagnosis of breast cancer was histologically confirmed in the remaining tissue. Blood samples were taken just before surgery, between 8 and 9 a.m. Blood was allowed to clot at room temperature and was centrifuged; the serum was separated and stored at — 30°C until the assay.
Tissue steroid assay
Intra-tissue steroid concentrations were measured according to the method of Van Landeghem et al. [11]. Aliquots of at least 0.8 g of pulverized tissue were homogenized in Tris-buffer (pH 7.4) and centrifuged at 100,000g for 30 min at 4°C. The cytosols were equilibrated with appropriate tritiated steroid tracers to enable correction of procedural losses. The cytosol samples were extracted with 4 vol of ethanol : acetone (1:1, v/v) and centrifuged at llOOg. The supernatants were evaporated and the residues defatted with 3 ml of 70% methanol in water. After centrifugation, the dried residues were redissolved in 25 μl of ethanol and 1 ml of Tris-buffer. Subsequently, the samples were ex-tracted 3 times with 3 ml of ether and the extracts were dried. The dried residues after ether extraction were resuspended in isooctane saturated with ethylene glycol. Oestradiol, testosterone and DHT were separated by partition chromatography on a celite column, imbued with propylene glycol/ethylene glycol 20:80 (v/v). Steroids were eluted with 3.5 ml of increasing amounts of ethyl acetate in isooctane: 3:97 (v/v) (DHT fraction); 18:82 (v/v) (testosterone fraction); 30:70 (v/v) (oestradiol fraction). The eluates were dried and left at 4°C until the radioimmunoassay.
Serum steroid assay
Three ml aliquots of serum were equilibrated with appropriate tritiated steroid tracers and extracted with 11 ml of ether. The ether extracts were dried and the steroids were separated on celite columns as reported for the tissue extracts.
Radioimmunoassay
Steroids, from tissue and serum, were measured by radioimmunoassay using commercial kits purchased from Biomerieux (Charbonnier le Bains, France). Tissue and serum samples of each patient were determined in the same kit. The sensitivities of the method in sera were 0.02 ng/ml for testosterone, 0.01 ng/ml for DHT, and 3.33 pg/ml for oestradiol. The sensitivities in tissue were 0.02 ng/g for testosterone and DHT and 10.0 pg/g for oestradiol. The practical sensitivities were determined by recoveries and by the quantity of tissue processed. Mean recoveries after the extraction and purification procedures for testosterone, DHT and oestradiol were 51.3, 39.0 and 78.8% in tissue and 80.0, 67.5 and 75.9% in sera, respectively. The intra-assay coefficients of variation (CV) for testosterone, DHT and oestradiol were 7.3, 9.7 and 4.0% in tissue and 7.2, 7.9 and 3.1% in sera, respectively. The inter-assay CV in the sera were 10.0, 11.0 and 8.9% for testosterone, DHT and oestradiol, respectively.
The serum oestradiol sample of one patient was lost during the assay procedure. The testosterone value in one tumour was found unaccountably high (1219 pg/g). It was considered an outlier and was excluded from the calculation.
Statistical analysis
Since the distributional form of hormonal levels in tissue and in blood did not satisfy the normal distri-bution assumption, the original values were trans-formed into their natural ranks [12]. The association between hormones was computed by the rank corre-lation coefficient rs (Spearman’s correlation coefficient). The mean hormonal levels in tissue and in blood were compared by the Wilcoxon signed rank test for matched pairs. The probability values were two tailed.
Results
Oestrogen receptor concentrations were obtained from the clinical records of 30 patients. No correlation was found between oestrogen receptor content and steroid levels measured in tumours. Intratumour concentrations and blood levels of testosterone, DHT and oestradiol are reported in Table 1. Mean oestradiol and DHT concentrations were significantly higher in tissue than in blood (P < 0.0001). Testosterone levels in tissues were widely dispersed and did not significantly differ from those measured in blood. The mean tis-sue/serum ratio for testosterone was close to 1, whereas a mean 9-fold increase in the ratio was observed for oestradiol and a mean 3-fold increase for DHT.
Table 1. Median and min—max for intratumour concentrations and blood levels of testosterone, dihydrotestosterone and oestradiol in 34 postmenopausal breast cancer patients.
A weak correlation was found between tissue and plasma concentrations of oestradiol, DHT and testosterone (Table 2). The correlation between tissue and blood levels of the 3 steroids are reported in Tables 3 and 4, respectively. Oestradiol levels were not associ-ated with those of DHT, either in blood or in tissue, whereas they were associated with those of testosterone in blood but not in tissue. The two androgens were correlated in tissue and in blood; correlation coefficients were computed after exclusion of an abnormal tissue testosterone value in one sample.
Table 2. Spearman correlation coefficient between tissue and blood levels of testosterone, dihydrotestosterone and oestradiol in postmenopausal breast cancer patients.
Table 3. Spearman correlation coefficient between tissue levels of testosterone (T), dihydrotestosterone (DHT) and oestradiol (E2) in postmenopausal breast tumours n = number of observations.
Table 4. Spearman correlation coefficient between blood levels of testosterone (T), dihy-drotestosterone (DHT) and oestradiol (E2) in post-menopausal breast tumors.
Sex steroids concentrations in the two tumours from the same patient are reported in Table 5.
Table 5. Testosterone (T), dihydrotestosterone (DHT) and oestradiol (E2) levels in the two tumours from the same patient and in the blood.
Discussion
A major role of oestrogen in the hormonal promotion of breast cancer has long been postulated. Although convincing evidence of hyperoestrogenemia has never been obtained in women with breast cancer [13], a high tissue/plasma ratio of oestradiol has been observed in these patients by several investigators [2-5] and in the present report, which demonstrates that oestrogen level in blood is not representative of the intratumour concentration of the steroid.
Studies from our laboratory have indicated that androgen excess is the endocrine abnormality which characterizes breast cancer patients [14-25], and elevated testosterone levels have been found in breast cancer patients by most of the case-control studies reported in the literature [26-34], though not by all [35-38]. In patients with early breast cancer, hyperan-drogenemia was found to be associated with an increased risk of developing metastases [18,25]. In hyperandrogenic women with metastatic breast cancer, remission of metastases by oophorectomy was associated with normalization of androgen excess [14, 16, 39]. These findings suggest that tumour growth is androgen-dependent in a subgroup of patients. The results of a study from Simon et al. [40] support the androgen-dependence of some mammary tumours. The authors observed that testosterone at physiological concentration stimulated cellular growth in 4 out of 7 shortterm cultures of mammary cancer cells derived from postmenopausal patients. Cellular growth is also known to be enhanced by androgens in some breast cancer cell lines [41,42]. Finally, the observation that the mammary gland has the same embryological origin as the apocrine sweat glands of the skin is suggestive for a growth stimulatory effect of androgens on the breast, since apocrine epithelium is under the control of androgens [43,44]. Women with apocrine cysts of the breast are at increased risk of breast cancer, and apocrine metaplasia is often associated with hyperplastic and neoplastic alterations of breast epithelium [45-47].
Studies on androgen concentrations in mammary tumours have shown that levels of androstenediol, dehydroepiandrosterone and androstenedione were higher in the tumour than in the blood [3,7,8,10], whereas the tissue/plasma ratio of testosterone was found to be close to 1 in the two studies that measured the hormone [3, 10] and in the present report. It should be noted, however, that the tissue/plasma ratio of testosterone varies widely, from 0.05 to 5.0 in the paper of Vermeulen et al. [3], and from 0.3 to 4.0 in the present report. This means that testosterone levels in a subgroup of tumours are substantially higher than in the blood but in another subgroup are lower. Whether biological and clinical differences exist between the two subgroups is not known.
In the present report, a 3-fold increase in intratu-mour DHT concentrations over circulating levels was observed. This finding corroborates previous data of Mistry et al. [10], who found significantly higher levels of DHT in breast cancer tissue than in blood. Evidence of 5α-reductase activity in mammary tumours [48] suggests that at least a portion of intratumour DHT arises from testosterone conversion within the breast. Testosterone is the immediate and obliged precursor of DHT in the biosynthetic pathway of androgens, and a correlation between these two steroids can be expected in tissues where the conversion of testosterone to DHT occurs.
Mistry et al. [10] reported a highly significant posi-tive correlation between intratumour concentrations of testosterone and DHT, and, in the present study, a similar strong linear correlation between the two an-drogens could be observed after the exclusion of an outlier. The ability of breast tumours to metabolize testosterone in 5α-reduced metabolites depends on the degree of apocrine differentiation of the tumour, and apocrine differentiation is frequently present in breast tumours [48].
The reported evidence indicates that large amounts of androgens are present in breast cancer tissue and suggests that androgens may play a role in the regulation of cancer growth. The route by which androgen influences growth of mammary cancer is not known, but several mechanisms can be assumed. Binding to androgen receptors may be important in stimulating growth of androgen-dependent tumours. Androgen receptors were found in 50-90% of breast tumours [9,49], and were associated with longer survival in operable disease and with favourable response to hormonal therapy in advanced disease [49]. Interestingly, a strong positive correlation has been reported among the presence of androgen, oestrogen and progesterone receptors in breast cancer tissue [50,51], suggesting that sex steroids act in concert in the growth regulation of mammary tumours. In addition to the physiological linkage to androgen receptor, there is evidence that androgen, at a high concentration, competes for oestrogen receptors in human breast cancer [52], in breast cancer cell lines [53] and in DMBA-induced rat mammary carcinoma [54], thus eliciting oestrogenic effects on tumour growth. A third mechanism is local conversion to oestrogen. In situ aromatization of androgen is thought to be an important source of intratumour oestrogen production [55, 56], although some authors have emphasized local sulphatase activity as the major determinant of intratumoural oestrogen levels [3,57]. The aromatase enzyme complex has been detected in 50-70% of human breast cancers [55, 56], and aromatase activity was found to be higher in mammary tumours than in normal breast tissue [3] and in the adipose tissue around the tumour [58].
In conclusion, a regulatory effect of androgens on breast cancer growth is conceivable, whatever the mechanism of action. We suggest that androgen and oestrogen act in concert in the control of tumour growth, and, in our opinion, further studies on this topic should be developed to investigate the occurrence of androgen/oestrogen imbalance in breast cancer.
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