Hormonal aspects in the causation of human breast cancer: epidemiological hypotheses reviewed, with special reference to nutritional status and first pregnancy.

Epidemiology of breast cancer has identified early age at menarche, late first pregnancy, low parity and late menopause as risk factors, but in addition genetic factors, height, weight and living in western countries play a significant role. The international variation in incidence is almost exclusively due to non-genetic factors. Hypotheses in prevention-oriented research are reviewed: 1. obesity-related oestrogen production as a stimulus of the tumour in postmenopausal women; 2. nutritional status and energy expenditure during puberty and adolescence, developed for fertility and fecundity and extended later to breast cancer; 3. reproductive life during early adulthood, age at first pregnancy and its specific effects on breast tissues. The message of preventability of breast cancer is that mammary epithelial differentiation should come early. Our insight concerning events in puberty and early adulthood can be consolidated in one concept on the risk of extended proliferation of breast epithelium during early adulthood in the absence of full differentiation induced by pregnancy. The combined effects of Western-type nutrition, lack of exercise and Western-type women's emancipation sets the stage for breast cancer already at a young age. Since it is unlikely that emancipated women in affluent societies will return to the original life-style of getting pregnant as soon as it is biologically possible, a novel daring way of protection has to be considered. Could a "Breast Differentiation Pill" be developed to offer protection?

Aromatase and comparative response to its inhibitors in two types of endometrial cancer.

Aromatase activity (AA) was evaluated totally in 80 tumors collected from primary endometrial cancer (EC) patients. All patients were divided into cases belonging to the types I or II of EC (respectively, 50 and 30 observations). Samples of malignant endometrium from type II demonstrated inclination to the higher AA in comparison with type I samples; the difference reached level of statistical significance in non-smoking patients (p=0.02). Although no positive correlation was revealed between AA in EC tissue and percentage of cells with DNA damage in normal endometrium from the same patients, the rate of DNA damage (percent of comets, comet's tail average length, etc.) was higher in intact endometrium collected from patients with type II of the disease. In 19 tumor samples, CYP19 gene expression was evaluated by RT-PCR and level of mRNA signal demonstrated positive correlation with AA (R(s)=+0.63, p=0.05) in the whole this material. Of note, though, CYP19 mRNA expression was not revealed in six cases, and all of them belonged to the type I of disease. Finally, in 23 EC patients (15 with type I and 8 with type II of the disease) effects of 2 weeks treatment with letrozole (10 pts) and exemestane (13 pts) were evaluated in neoadjuvant setting. Although diminishing of endometrial M-echo signal and the increases in FSH and LH concentration after treatment were more pronounced in type I patients, decrease in tumor PR content (p=0.04) was more revealing in patients with type II of EC; besides, the decreases in AA in tumor tissue by the end of treatment were noted predominantly in patients with lower body weight (BMI <27.5). Thus, although type II of EC is frequently considered as hormone-independent, increased ability of this type of the tumor to estrogen biosynthesis (at CYP19 gene and protein level) may lead to the reconsideration of such conclusion and warrants further investigation. The search of possible ethnic differences in AA and in the biologic response to aromatase inhibitors in EC can be of importance too.

The role of luteinizing hormone in the pathogenesis of hyperadrenocorticism in neutered ferrets.

Four studies were performed to test the hypothesis that gonadotrophic hormones, and particularly luteinizing hormone (LH) play a role in the pathogenesis of ferrets: (I) adrenal glands of ferrets with hyperadrenocorticism were studied immunohistochemically to detect LH-receptors (LH-R); (II) gonadotrophin-releasing hormone (GnRH) stimulation tests were performed in 10 neutered ferrets, with measurement of androstenedione, 17alpha-hydroxyprogesterone and cortisol as endpoints; (III) GnRH stimulation tests were performed in 15 ferrets of which 8 had hyperadrenocorticism, via puncture of the vena cava under anesthesia; and (IV) urinary corticoid/creatinine (C/C) ratios were measured at 2-week intervals for 1 year in the same ferrets as used in study II. Clear cells in hyperplastic or neoplastic adrenal glands of hyperadrenocorticoid ferrets stained positive with the LH-R antibody. Plasma androstenedione and 17alpha-hydroxyprogesterone concentrations increased after stimulation with GnRH in 7 out of 8 hyperadrenocorticoid ferrets but in only 1 out of 7 healthy ferrets. Hyperadrenocorticoid ferrets had elevated urinary C/C ratios during the breeding season. The observations support the hypothesis that gonadotrophic hormones play a role in the pathogenesis of hyperadrenocorticism in ferrets. This condition may be defined as a disease resulting from the expression of LH-R on sex steroid-producing adrenocortical cells.

Insulin resistance, its consequences for the clinical course of the disease, and possibilities of correction in endometrial cancer.

OBJECTIVES: To study the frequency of insulin resistance (IR) in endometrial cancer patients, its relation to the clinical course of the disease and DNA damage, and to evaluate possible approaches to the pharmacological correction of IR in the patients studied. METHODS: The signs of insulin resistance syndrome and its association with the clinical and pathological features of the disease and DNA damage in somatic cells (micronucleus frequency in peripheral blood lymphocytes) and endometrial normal and tumor tissue (alkaline unwinding) were determined in 99 endometrial cancer patients. RESULTS: The frequency of insulin resistance syndrome counted on the basis of fasting plasma glucose and insulin concentrations according to Duncan et al. is equal to 0.35 (95% CI 0.24-0.46), or 35%, in endometrial cancer patients who do not have a history of diabetes mellitus. Patients with well- or moderately differentiated endometrial adenocarcinomas (mostly type I) had statistically significantly higher basal and stimulated plasma insulin and C-peptide concentrations than patients with poorly differentiated endometrial adenocarcinomas or rarely encountered tumors of the endometrium (primarily type II). Interestingly, the level of fasting insulinemia positively correlates with disease stage and with local and regional tumor dissemination only in the group of patients with well- or moderately differentiated endometrial adenocarcinomas. On the other hand, hyperinsulinemia and other hormonal-metabolic disturbances typical of insulin resistance syndrome do not increase the probability of DNA damage of somatic cells (according to the data of micronucleus test). In addition, no association between hormonal-metabolic disturbances and the degree of DNA unwinding in tumor and visually unchanged endometrium was found. CONCLUSION: Thus, insulin resistance/hyperinsulinemia is associated with a more aggressive course of the disease in certain groups of the patients but--in contrast to excessive estrogenic stimulation--does not result in increased genotoxic damage in tumor and normal tissues. The data obtained once more confirm the need for treatment and prevention measures aimed at correcting hormonal-metabolic disturbances in endometrial cancer patients and groups at risk of this disease. Such an approach might include use of antidiabetic biguanides, thiazolidinediones (glitazones), and statins.

Tumor estrogen content and clinico-morphological and endocrine features of endometrial cancer.

OBJECTIVES: To compare estrogen concentrations in endometrial cancer tissue with those in macroscopically normal endometrium and with certain morphological characteristics of the tumor and endocrine parameters in patients. METHODS: The estradiol content was evaluated by radioimmunoassay after homogenization and extraction in 78 adenocarcinomas (61 from postmenopausal patients). RESULTS: Higher concentrations of estradiol in tumor tissue samples than in macroscopically normal endometrium were found in patients of both reproductive and postmenopausal age. This difference was the same in patients with either endometrial carcinoma type I or type II. No association between tumor steroid receptor levels, estradiol concentrations in blood serum, and timing of menopause with intratumoral estradiol contents was discovered. Estradiol concentrations in tumor tissues correlated positively with the clinical stage of disease and rate of tumor invasion (in patients with peripheric/lower type of fat topography), and negatively with tumor differentiation stage (in patients with central/upper type of fat topography) and the percentage of intact double-stranded DNA in normal endometrium. CONCLUSIONS: Tumor estrogen content in endometrial cancer has clinical significance that is modified in the presence of certain endocrine characteristics related to insulin resistance. The role of local estrogen production (aromatase activity) in this setting deserves special study.

Effect of tibolone (Org OD14) and its metabolites on aromatase and estrone sulfatase activity in human breast adipose stromal cells and in MCF-7 and T47D breast cancer cells.

Tibolone (Org OD14) is a synthetic steroid used for post-menopausal hormone replacement therapy (HRT). Since HRT might increase breast cancer risk, it is important to determine the possible effects of tibolone on breast tissues. Tibolone and its metabolites Org 4094, Org 30126 and Org OM38 have been reported to inhibit estrone sulfatase activity in MCF-7 and T47D breast cancer cell lines, which suggest beneficial effects on hormone dependent breast cancer by reducing local production of free estrogens. Breast adipose stromal cells (ASCs) contain aromatase activity-an obligatory step in the biosynthesis of estrogens-and possibly contain sulfatase activity. We investigated the effects of tibolone, its metabolites and the pure progestin Org 2058 on PGE(2)-stimulated aromatase activity and on sulfatase activity in human ASC primary cultures and on sulfatase activity in MCF-7 and T47D cell lines. In MCF-7, tibolone and metabolites, but not Org 2058, were found to inhibit sulfatase activity. In T47D, tibolone inhibited sulfatase only at 10(-6)M, although weakly. ASC had high sulfatase activity, which was inhibited by 10(-6)M of tibolone, Org 4094 and Org 30126, but not by Org OM38 or Org 2058. Surprisingly, aromatase activity in ASC was increased by both tibolone and Org 2058 at 10(-6)M. As ligand binding assay results and immunohistochemistry indicated the absence of progesterone and estrogen receptors in ASC, these effects on aromatase and sulfatase activity in ASC likely take place by other routes. Because tibolone and its metabolites inhibit sulfatase activity, and because tibolone only increases aromatase activity at a high concentration, we conclude that effects of tibolone on the breast are probably safe.

Artifact formation due to ethyl thio-incorporation into silylated steroid structures as determined in doping analysis.

Trimethylsilylation of target substances in a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide and ethanethiol is frequently applied for the application of gas chromatography-mass spectrometry (GC-MS) in steroid analysis. However, artifacts were formed when using this mixture to silylate the steroids androsterone and etiocholanolone obtained from a urine matrix. The artifacts were identified as ethyl thio-containing products of the respective trimethylsilyl derivatives. The conversion of the studied products increased slowly as a function of time, was dependent on the presence of the urine matrix and was significantly accelerated by adding diethyl disulfide to the reagent before incubation. Also ethyl thio-incorporation into testosterone and epitestosterone was established. A mechanism for ethyl thio-incorporation is proposed. The conversion achieved after 120-h sample storage at room temperature was insufficient to significantly influence the analysis of androsterone and etiocholanolone under the studied conditions. However, the results provide fundamental insight into the mechanism of silylation and the occurring side-reactions. Moreover, when investigating the formation of new metabolites, the ethyl thio-incorporation can lead to misinterpretation.

TP53 mutations in human meningiomas.

Overexpression of p53 has been reported to play a role in the development of neoplasms of the central nervous system. Meningiomas are generally benign intracranial tumors originating from the meninges. Overexpression of the p53 protein in meningiomas and an association with histological type and recurrence has been reported. Mutation of the TP53 gene leads to a more stable p53 protein in quantities high enough for detection by immunohistochemistry. In the search for these mutations the core domain of the TP53 gene of meningiomas has been analyzed. Only a very low incidence of mutations was reported. The apparent discordance between overexpression of p53 protein and TP53 gene mutations may be explained by mutations located outside the core domain. This issue was addressed in the present study. All 11 exons of 17 meningiomas were analyzed for DNA alterations by PCR single-strand conformation polymorphism (PCR-SSCP) analysis with subsequent sequencing. PCR-SSCP analysis showed a various number of band shifts and nucleotide alterations, caused either by alterations in the flanking introns or common polymorphisms (codon 36 and 72). The allele frequencies of the polymorphisms found in this small population of tumors resemble the frequencies reported in the literature. In addition, three nucleotide changes located in introns 2, 3 and 7 were found in 11, 3 and 4, respectively, of 17 specimens. Based on this study and on reports by others we conclude that it is not very likely that TP53 mutations are involved in the etiology of meningiomas.

Lipids and apoproteins in relation to participation in organized sport activities and pubertal development in boys.

This study considers changes in the plasma lipid and apoprotein profiles of boys in relation to participation in organized sport activities and to testosterone (T) levels. Fifty boys, aged 9.9 ± 0.6 years (mean ± S.D.), participated in the study. During a 3 year follow-up, the following measurements were taken twice a year: stature, weight, and skinfolds. Blood samples for lipids and apoproteins and sex hormone levels, and information on participation in sport activities were also obtained. No relationship was found between participation in organized sport activities and high density lipoprotein cholesterol (HDL-C) or apoprotein A-I (apo A-I). The changes of the profile over time in more active boys (participation rate > 3 hr/wk) were similar to those of less active boys (participation rate < 1 hr/wk) (MANOVA, repeated measures, not significant.) Consistent relationships between sex hormones and lipids and apoproteins were restricted to T with total cholesterol (TC), HDL-C, and apo A-I, respectively. The common variance ranged from 5.8% (rT,TC ) to 18.5% (rT,HDL-C ) (P ⩽ 0.05.) When the boys who reached advanced puberty during the follow-up period (n = 21) were studied apart from those who did not (n =29), differences were found in TC, apo A-I, and HDL-C, TC decreased from 4.6 ± 0.65 to 4.3 ± 0.58 mmol/l in the more advanced pubertal boys, and increased from 4.6 ± 0.90 to 4.8 ± 0.79 mmol/l in the others; apo A-I decreased from 185 ± to 28.3 to 156 ± 20.4 mg/dl and from 179 ± 20.6 to 176 ± 27.7 mg/dl, respectively (MANOVA, repeated measures, P ⩽ 0.05) HDL-C was lower in advanced pubertal boys at the end of follow-up (1.4 ± 0.33 and 1.7 ± 0.38 mmol/l, respectivel; P ⩽ 0.05). The lack of a relationship with regular physical activity may be due to the high levels of HDL-C and apo A-I at the begining of the study. On the other hand, the effect of the increasing T levels on HDL-C and apo A-I may have overwhelmed the presumed effect of regular physical activitiy. © 1995 Wiley-Liss, Inc.

The onset of puberty and the availability of sex steroids in female gymnasts.

Plasma levels of gonadotropins, (sex) steroids, SHBG bound fractions of 17ß-estradiol (E2) and testosterone (T), and sex-hormone-binding globulin (SHBG) were compared at the onset of puberty in female gymnasts (n = 7) and two groups of schoolgirls with similar anthropometric characteristics. Ten schoolgirls were matched to the gymnasts on the basis of a sum of skinfolds and the waist/hip ratio, while 12 other girls were selected on the basis of the stature and bicristal breadth of the female gymnasts. All girls were in the second stage of breast development (M2) and were classified as being in the first stage (M1) 6 months earlier. When female gymnasts were compared to nonathletic schoolgirls with similar physical characteristics at the same stage of early puberty, the former had significantly lower levels of plasma luteinizing hormone (LH), total and available E2, and T. In conclusion, female gymnasts have significantly lower LH, E2, and T plasma levels than nonathletic schoolgirls in early puberty. The E2 and T plasma levels in early female gymnasts are not related to the individual physical characteristics, i.e., fat mass, short stature, or small bicristal breadth. © 1993 Wiley-Liss, Inc.

Effects of anaesthesia and manual restraint on the plasma concentrations of pituitary and adrenocortical hormones in ferrets.

Two experiments were carried out to investigate the effect of sampling techniques on the plasma concentrations of pituitary and adrenocortical hormones in ferrets (Mustela putorius furo). In the first experiment blood was collected on two occasions from 29 ferrets which were either manually restrained or anaesthetised with isoflurane. In the second experiment eight intact ferrets were fitted with jugular catheters and blood was collected on four occasions, just before and as soon as possible after they had been manually restrained or anaesthetised with medetomidine or isoflurane; blood was also collected 10 and 30 minutes after the induction of anaesthesia. Medetomidine anaesthesia had no effect on the plasma concentrations of pituitary and adrenocortical hormones. Isoflurane anaesthesia resulted in a significant increase in the plasma concentration of alpha-melanocyte-stimulating hormone (alpha-MSH) directly after the induction of anaesthesia. Manual restraint resulted in a significant increase in the plasma concentrations of cortisol and adrenocorticotrophic hormone (ACTH) and a decrease in the plasma concentration of alpha-MSH.

[Marius Tausk (1902-1990), influential endocrinologist and producer of medicines; a retrospect to mark the centenary of his birth]. / Marius Tausk (1902-1990), invloedrijk endocrinoloog en producent van geneesmiddelen; terugblik bij zijn honderdste geboortedag.

Marius Tausk, born in Sarajevo, studied medicine at Graz in Austria. In 1926, he attended a socialist youth rally in Amsterdam. Whilst there, he met Prof. Ernst Laqueur, a pharmacologist in Amsterdam, who offered him a position at Organon, a newly-founded pharmaceutical company in Oss, the Netherlands. He remained in the Netherlands and became the driving force behind this innovative company. Tausk made many contributions to new developments across the field of endocrinology, including the discovery of the adrenal steroids (together with T. Reichstein, Nobel Prize Laureate in 1950), and the development of oral contraceptive pills. He was astute enough to quickly patent the first corticosteroids. He could quickly extract the essential elements from an information source and disseminate this in five languages. His sharp judgment brought him friends and admirers, yet also those who feared him. In 1937 he was appointed as a private lecturer at the Medical Faculty of Utrecht University and in 1956 he was made Special Professor of Theoretical Endocrinology. He was awarded a number of scientific distinctions including two honorary doctorates. In 1949 he was honoured with the Knighthood of the Order of The Netherlands Lion.

Is aromatase cytochrome P450 involved in the pathogenesis of endometrioid endometrial cancer?

Prospectively, the relationship between androgen levels in the utero-ovarian circulation, aromatase activity in endometrial and body fat tissue, and the presence or absence of endometrioid endometrial cancer was studied in postmenopausal women. In 43 women with endometrioid endometrial cancer and 8 women with a benign gynecological condition, a hysterectomy with bilateral salpingo-oophorectomy was performed. Using tritium water-release assays, aromatase activities in endometrial and body fat tissue were determined and related to the steroid levels from the peripheral and the utero-ovarian venous circulation (estradiol, androstenedione, testosterone) and to the presence or absence of endometrial cancer. Significant aromatase activity was found in both benign and malignant endometrial tissue samples. Aromatase activity in samples of endometrial tissue and in samples of body fat did not correlate with steroid levels in peripheral or utero-ovarian venous blood. Aromatase activity in samples of benign or malignant endometrium did not differ. Remarkably, in four women with mainly poorly differentiated endometrial cancer, very high aromatase activity was found in endometrial tissue. It is likely that multiple pathogenetic pathways exist that eventually lead to the formation of endometrioid endometrial cancer. The local availability of androgens and the finding that aromatase activity is present in both endometrial cancer and benign endometrial tissue support the hypothesis that aromatase activity in the endometrium may play a role in malignant transformation by converting androgens into mitogenic estrogens in the endometrial tissue.

Progesterone receptor, bc1-2 and bax expression in meningiomas.

Meningiomas are generally benign central nervous system neoplasms, which frequently express progesterone receptor (PR) and only rarely express the estrogen receptor (ER). For breast cancer, a relation between steroid hormone receptors and proteins involved in the apoptotic process has been described. For meningiomas, the exact relation between PR and these proteins is not known. In this study, ER, PR, bcl-2 and bcl-2-associated x protein (Bax) expression levels were determined in meningioma cytosols. As a reference for our experimental conditions, we also determined these proteins in breast cancer cytosols. PR and ER were determined with a ligand-binding assay and scatchard-plot analysis. The expression levels of the anti- and pro-apoptotic proteins, bcl-2 and Bax, respectively, were determined by immunoblotting. In 65% of the meningioma, bcl-2 expression was found in variable amounts. In contrast to breast cancer, a significant negative association between PR and bcl-2 was found (P < 0.01). Bax expression appeared constitutive, not related to PR, and 2.6 times higher than breast cancer. As both PR and bcl-2 appear positively associated with prognosis, the negative relationship between bcl-2 and PR found in this study might have some biological and clinical significance.

Role of progestins with partial antiandrogenic effects.

An experts' meeting on the 'Role of progestins with partial antiandrogenic effects' was held in Berlin from January 19 to 22, 2001. The meeting was chaired by Dr R. Sitruk-Ware (New York, USA) and participants included Ms F. Fruzzetti (Pisa, Italy), J. Hanker (Trier, Germany), J. Huber (Vienna, Austria), F. Husmann (Bad Sassendorf, Germany), S. O. Skouby (Copenhagen, Denmark), J. H. H. Thijssen (Utrecht, The Netherlands), and R. Druckmann (Nice, France). The present paper reports the conclusions of the meeting. However, the publication of the Women's Health Initiative study, which appeared after the meeting, led to additional comments and revisions.