Richard J. Santen

Aromatase activity in primary and metastatic human breast cancer

Aromatase activity was measured in 104 primary and 24 metastatic breast cancer patients. The assay employed quantitates the production of 3H water release from 1β‐[3H] androstenedione after aromatization. Of 104 human primary breast tumors studied, 64 contained measurable aromatase activity, ranging from 5–70.5 pmol estrone formed/g protein/hour. In primary breast cancers there was no difference in levels of aromatase activity when analyzed by menstrual status or age by decade. Aromatase activity was similar in small and large primary tumors. The median aromatase activity of primary breast tumors (8.6 pmol/g/h) was similar to that found in metastatic breast cancer deposits (12.0 pmol/g/h). Aromatase activity did not correlate with either estrogen (ER) or progesterone (PR) receptor concentration in the tissues assayed. In this regard there were 33 ER− PR− tumor biopsies. Twelve of these 33 tumors contained aromatase activity greater than 10 pmol/g/hour. Cancer 59:779‐782, 1987.

Enzymatic Control of Estrogen Production in Human Breast Cancer: Relative Significance of Aromatase versus Sulfatase Pathways

One-third of the cases of breast cancer in postmenopausal women are hormone-dependent and the lesions regress upon treatment with antiestrogens or inhibition of estrogen biosynthesis. In these patients, estrogens are synthesized in extraglandular tissues from adrenal precursors and re-enter plasma to produce estrone levels of 52 +/- 6.5 pg/ml (mean +/- SEM) and estradiol concentrations of 13.1 +/- 0.7 pg/ml. However, the fact that the levels of estrogen in breast tumor tissue are an order of magnitude higher than plasma levels suggested the possibility of in situ estrogen production. To address this possibility, we measured several enzymes involved in estradiol biosynthesis in human tumors. Forty-eight of 61 tumors contained aromatase (estrogen synthetase) activity ranging from 5-80 pg/gm protein per hour. By comparison, the levels of estrone sulfatase were 10(6) higher, ranging from 0.8-125 micrograms/gm protein per hour. Because the sulfatase enzyme was of lower affinity (i.e., Km = 27 microM) than that of aromatase (i.e., 0.027 microM), the amount of estrogen formed under conditions of similar substrate concentrations was compared and found to be 10-fold higher via the sulfatase enzyme. In 41 additional tumors, the 17 beta-hydroxysteroid dehydrogenase enzyme, catalyzing the conversion of estrone to estradiol, was uniformly present. To test the biologic relevance of the estrone sulfate to estrone to estradiol pathway, estrogen-dependent nitrosomethylurea rat mammary tumors were grown in soft agar in the presence of estrone sulfate. Concentrations of estrone sulfate of 10(-6) microM significantly (p less than 0.01) stimulated colony formation in this system in which 75.5-98.6% of estrone sulfate was converted to estrone and 0.2 to 6% to estradiol. These data support the hypothesis that mammary carcinomas can synthesize estradiol in situ from circulating estrogen precursor and that local conversion is biologically important. On the basis of comparative data, the estrone sulfate to estrone to estradiol pathway is quantitatively more important than that involving androstenedione to estrone to estradiol.

Androgen priming and chemotherapy in advanced prostate cancer: evaluation of determinants of clinical outcome.

We conducted a randomized clinical trial in men with stage D2 prostate cancer to test whether androgen priming potentiates the efficacy of cytotoxic chemotherapy. Eighty-five men with progressive prostate cancer refractory to orchiectomy were treated continuously with aminoglutethimide and hydrocortisone to lower adrenal androgen secretion and were administered cyclic intravenous (IV) chemotherapy. The patients were randomized to receive either androgen priming or no additional treatment for three days before and on the day of chemotherapy. Median duration of follow-up was 43 months. Response rate (remission plus disease stabilization) was not significantly different between the stimulation and control arm when the analysis was restricted to evaluable patients (79% v 73%, respectively) or when it was extended to all patients (46% v 61%). Median duration of response was similar for the stimulation and control arm (9 and 10 months, respectively). Median survival was 10 months in the stimulation and 15 months in the control group (P = .0047). The androgen sensitivity of the tumors was supported by the greater toxicity in the stimulation arm associated with androgen administration. Factors found to be independently associated with improved clinical outcome included a high Karnofsky score and hematocrit, long duration of response to the initial castration, and normalization of an elevated serum acid phosphatase on treatment. We conclude that in this group of patients with advanced disease, androgen priming does not potentiate the efficacy of chemotherapy and is actually associated with a worse outcome. Furthermore, our data emphasize the heterogeneity of biologic behavior of prostate cancer.

Medical adrenalectomy using aminoglutethimide and dexamethasone in advanced breast cancer

Twelve women with advanced breast carcinoma were given aminoglutethimide (up to 2 gm q.d. p.o.), in addition to dexamethasone (1.0–3.0 mg daily) and Florinef (0.1 mg b.i.w.) in order to produce a medical adrenalectomy. Adequacy of adrenal blockade was assessed by measuring A.M. plasma and 24‐hour urinary free cortisol by specific competitive protein binding techniques. Seven of 9 patients who had received an adequate trial had constant suppression of adrenal cortical function for at least a period of 8 weeks. Escape from blockade, when observed, was attributed to the rapid metabolism of dexamethasone induced by aminoglutethimide, demonstrated by determining the half‐life of 3H‐dexamethasone before and during treatment. Three patients with soft tissue involvement demonstrated a 1‐A response (12+, 5+ 6 months). Three patients with bone involvement showed a 0‐A response (7, 5, and 4 months). Lethargy and allergy to aminoglutethimide prevented an adequate trial in two patients. A third patient who was adequately suppressed for 7 weeks died before the 8‐week trial period. Minor side effects attributable to aminoglutethimide (nystagmus, ataxia, lethargy, skin rash) were seen frequently but abated with reduction in dosage. No Cushingoid side effects related to dexamethasone administration were noted.

The efficacy of CGS 20267 in suppressing estrogen biosynthesis in patients with advanced stage breast cancer.

The pharmacologic inhibition of aromatase activity has been the focus of clinical trials in patients with advanced stage breast cancer. Recent developments with imidazole compounds that inhibit aromatase activity suggest their clinical use as potent inhibitors of estrogen biosynthesis in postmenopausal breast cancer patients. In this Phase I, open-label, dose-range finding study, we examined the inhibitory potency of CGS 20267 on blood and urine levels of estradiol, estrone and estrone sulfate in 8 patients with metastatic breast cancer. Studies included evaluation of adrenal and thyroid function to look for evidence of general hydroxylase inhibition at dose levels effective for aromatase blockade. Patients were administered CGS 20267 at doses of 0.1 and 0.25 mg, once a day in ascending doses over a 12-week period. Preliminary data reveal that CGS 20267 elicits a striking suppression in plasma estradiol, estrone and estrone sulphate which was observed in some patients as quickly as within 24 h of the first dose. Estrogen suppression of over 90% was achieved within 2 weeks of therapy. No alterations in either baseline or ACTH (cortrosyn) stimulated cortisol and aldosterone levels were observed through the 12 weeks of therapy. In addition, 24 h urine sodium and potassium values were not appreciably altered during therapy. We conclude that CGS 20267 is a potent, specific inhibitor of estrogen biosynthesis in postmenopausal patients with metastatic breast cancer and effectively reduces blood and urine estrogens to undetectable levels.

Plasma estrone-sulfate assessment of reduced estrogen production during treatment of metastatic breast carcinoma

Highly sensitive and specific estrogen assays are required to monitor the hormonal effects of surgical adrenalectomy or pharmacologic estrogen suppression in postmenopausal women with breast carcinoma. Because the levels of plasma estrone-sulfate are 10-fold higher than its unconjugated counterpart, we developed a radioimmunoassay for estrone-sulfate to quantitate the minimal estrogen concentrations expected under conditions of endocrine gland ablation. After establishing normal ranges, we compared plasma estrone- sulfate levels and urinary conjugated estrone basally and after surgical adrenalectomy or aminoglutethimide (estrogen suppression) therapy in 23 postmenopausal women with breast carcinoma. In response to either therapy, the plasma levels of estrone-sulfate fell by 63.5-79.2% (p less than .01) and conjugated urinary estrone by 85-94.5% (p less than .01) in all study days over a 12-week period. Correlation analyses yielded r values of 0.77-0.94 between conjugated plasma and urinary estrone concentrations in the surgical adrenalectomy and aminoglutethimide-treated groups, respectively. No significant differences in estrone-sulfate levels were observed when comparing spontaneously menopausal and surgically castrate patients.

Estrone sulfate: A potential source of estradiol in human breast cancer tissues

SummaryLocal formation of estradiol in human breast tumors could provide a more important source of estrogen than is delivered from plasma. Prior studies have suggested that estrone is primarily synthesized from estrone sulfate. The enzyme 17β-hydroxysteroid dehydrogenase (HSD) would be required to convert estrone to estradiol.This study characterized HSD in 1000 × g supernatants from human breast tumors. Estradiol synthesis was linearly related to tissue concentration or time over the range studied. Cofactor requirements varied with estrone concentration. High and low affinity sites were found in 50% of tissues studied, while the remainder contained only low affinity sites. Screen assays showed measurable activity in all 42 samples tested. This activity ranged from 0.73−>100 nmol estrone synthesized/g protein/hr, with a median activity of 5.9 nmol/g/hr.We evaluated the biological relevance of the sulfatase-HSD pathway by testing the ability of estrone sulfate to stimulate colony formation in soft agar cultures of nitrosomethylurea-induced rat mammary tumors. The maximally effective concentration ranged from 10−7 to 10−4 M. Significant stimulation of colony formation was observed in 7 of 8 experiments. The estrone sulfate stimulation pattern was similar to that previously observed with estradiol. Of the3H-estrone sulfate added to the dishes, 20–98% was recovered as estrone and 0.2–6% as estradiol. These studies suggest that the requisite enzymes are present in human breast tumors for conversion of estrone sulfate to estradiol, and that this pathway may be biologically significant.

Medical adrenalectomy with aminoglutethimide: clinical studies in postmenopausal patients with metastatic breast carcinoma.

The use of adrenalectomy and hypophysectomy in the management of postmenopausal patients with metastatic breast carcinoma is reserved for highly selected patients. As an alternate approach, a pharmacologic method of inhibiting adrenal cortical secretion was developed which consisted of the daily administration of 1000 mg of aminoglutethimide to block steroidogenesis and either dexamethasone (2.0–3.0 mg/day) or hydrocortisone (40–60 mg/day) as replacement glucocorticoid. This regimen markedly suppressed plasma levels of DHA-S, androstenedione, estrone, and estradiol, and urinary levels of aldosterone. Of 50 patients treated, 19 (38%) demonstrated either a complete (8/19) or a partial (11/19) objective disease remission which lasted for 18.05 ± 3.1 months (mean ± SEM). In 10 (20%) patients, there was stabilization of disease (7.8 ± 1.2 months), accompanied by symptomatic relief of bone pain in six (12%). There was disease progression in 20 (40%) patients. The acute side effects of aminoglutethimide therapy were significant and consisted of transient lethargy (41.5%) and a cutaneous rash (35.8%). Chronic toxicity was negligible. The medical adrenalectomy regimen of aminoglutethimide plus glucocorticoid offers a suitable alternative to surgical adrenalectomy or hypophysectomy in the management of postmenopausal patients with metastatic breast carcinoma.

Mechanism of Action of Narcotics in the Production of Menstrual Dysfunction in Women

The ability of morphine to block ovulation in animals prompted investigation of the frequency and mechanisms of menstrual abnormalities in women addicted to narcotic analgesics. Menstrual histories obtained from 76 former heroin addicts receiving daily methadone maintenance revealed that more than one-half of these women had experienced menstrual abnormalities while taking heroin or methadone. In order to determine the specific physiologic effects of narcotic analgesics on reproductive function, detailed endocrinologic studies were carried out in seven of these patients who complained of amenorrhea or irregular menses while receiving methadone. Four of the seven women manifested abnormalities of the control of gonadotropin secretion. Three of these four failed to exhibit cyclic gonadotropin release, as evidenced by an absence of increased levels of follicular phase follicle-stimulating hormone, midcycle gonadotropin peaks or luteal phase progesterone increments. In the fourth patient a prolonged follicular phase (30 days) of the menstrual cycle was detected. One of these four patients also had low basal gonadotropin levels and failed to exhibit luteinizing hormone increments greater than control levels in response to ethinyl estradiol (positive feedback). The remaining three women exhibited normal patterns of gonadotropin secretion during the observation period. In these women, menstrual bleeding occurred in response to withdrawal from luteal phase (10 to 20 ng/ml) progesterone levels and to exogenous ethinyl estradiol, suggesting normal uterine responsivity to progesterone and estrogen. Although not documented, it is likely that oligo-ovulation was the cause of the irregular menses in these three patients. Amenorrhea is commonly associated with methadone ingestion or heroin addiction and appears to be related to an alteration of the hypothalamic mechanisms controlling gonadotropin secretion. Tolerance to these effects of methadone may develop after chronic ingestion.

Kinetic, hormonal and clinical studies with aminoglutethimide in breast cancer

Approximately one‐third of patients with metastatic breast carcinoma respond to surgical ablative therapy but the morbidity associated with these procedures has limited their use to highly selected patients. Consequently, a chemical method of adrenal suppression was developed using a potent inhibitor of adrenal steroid synthesis, aminoglutethimide, in combination with a synthetic glucocorticoid, dexamethasone. While this regimen effectively blocked adrenal function, it was complicated by a drug interaction in which aminoglutethimide accelerated the metabolism and reduced the bioavailability of dexamethasone. To overcome this problem, a new regime using aminoglutethimide and hydro‐cortisone, a glucocorticoid less susceptible to altered metabolism, was developed. Kinetic studies confirmed that aminoglutethimide does not interact with hydrocortisone to alter its rate of metabolism. Hormone measurements established that 1000 mg of aminoglutethimide and 40 mg of hydrocortisone daily suppressed DHA‐sulfate, androstenedione, estrone, estradiol and aldosterone to a greater extent than the prior protocol using aminoglutethimide and 2–3 mg of dexamethasone. Patients experienced objective tumor regression with equal frequency while receiving the aminoglutethimide‐hydrocortisone regimen or aminoglutethimide and dexamethasone and the overall rate of response in 50 evaluable patients was 38%. Side effects occurred frequently in the first few weeks of treatment but disappeared nearly uniformly thereafter. The present aminoglutethimide‐hydrocortisone regimen is simple, non‐toxic, effective in inhibiting estradiol synthesis and capable of inducing tumor regression as frequently as previously reported with adrenalectomy.