Free fatty acid concentrations correlated with the available fraction of estradiol in human plasma.

A physiological in vivo increase of plasma free fatty acid concentration after an overnight fast was found to be accompanied by a rise of the non-protein bound estradiol fraction. A similar increase was observed after lipase activation by the i.v. injection of 500 IU heparin in 5 healthy non-fasting subjects. In vitro studies showed a direct relationship between non-protein bound estradiol and the concentration of linoleate, linolenate, and arachidonate both in undiluted serum and in Ringer's solution containing human serum albumin (45 g/liter). Moreover, the estradiol sex hormone binding globulin complex bound to a solid concanavalin A-Sepharose matrix was markedly dissociated by oleate and even more by linoleate, linolenate, or arachidonate. These results suggest that physiological diurnal elevations in plasma free fatty acids which are amplified by high fat consumption, obesity, and stress may imply major proportional increases of available estradiol, exerting a promotional effect on breast and endometrial cancer over the years.

MAM-6 antigen, a new serum marker for breast cancer monitoring.

Almost all carcinomas contain a cell surface antigen, MAM-6, which has been defined by several monoclonal antibodies, including 115D8 (Hilkens et al., Int. J. Cancer, 34: 197-206, 1984). A quantitative sandwich radioimmunoassay, using 115D8 as catcher and as tracer antibody, has been developed to detect MAM-6 in serum. To quantitate the MAM-6 level, pooled human milk was used as a standard, and arbitrary units were chosen. Less than 5% of the sera of apparently healthy individuals contained more than 5 units/ml. In sera of patients with benign breast lesions, the same low levels were detected. However, concentrations over 5 units/ml were found in 24, 21, 43, and 79% of the sera of patients with pathological Stages I, II, III, and IV breast cancer, respectively. MAM-6 levels were also increased in almost all sera tested from patients with advanced stages of ovarian carcinoma, but in a low percentage of sera from patients with other advanced cancers. A longitudinal study was carried out to test the MAM-6 assay as clinical marker to monitor the therapeutic response of breast cancer. Increasing or decreasing MAM-6 serum levels correlated in 93% of the cases with breast cancer progression or regression, indicating that the assay can be used to monitor the course of the disease during therapy. In some breast cancer patients, elevated MAM-6 levels were observed prior to any clinical indication of tumor recurrence.

Thyrotropin dependent and independent thyroid cell lines selected from FRTL-5 derived tumors grown in nude mice.

FRTL-5 cells were used to set up a thyroid tumor model system in C3H nu/nu mice. FRTL-5 tumors could be grown in nude mice provided serum TSH levels were elevated. Persistent TSH elevation was obtained by administration of Na131I, rendering the mice hypothyroid. After 4 weeks FRTL-5 cells were injected sc resulting in tumor growth within 2 weeks in eight out of eight mice. Although the tumors showed an apparently undifferentiated histology, lacking normal follicular structures, they were functional since the tumors were capable of concentrating [131]iodine, as demonstrated by nuclear imaging. From one of the tumors a new cell line was isolated (FRTL-5/T) that, like the parental FRTL-5 cell line, was TSH dependent for growth. In a control group of six euthyroid nude mice FRTL-5 tumor growth could not be obtained with one exception. After 3 months one animal developed a small tumor that grew rapidly thereafter. This tumor was easily transplantable in other euthyroid nude mice, showed an undifferentiated histology, and was nonfunctional, as it could not concentrate [131]iodine. From this tumor two cell lines were derived: one cultured in the presence of TSH (FRTL-5/TP) and one in the absence of TSH (FRTL-5/TA). Both cell lines were found to be TSH independent for growth. The cell lines were analyzed for TSH responsive functions and TSH receptor expression. Responsiveness to TSH in FRTL-5/T and the parental FRTL-5 cell line were similar for most thyroid specific functions tested. However, FRTL-5/T was less sensitive than FRTL-5 for TSH induced [3H]thymidine incorporation. Both cell lines had two classes of TSH binding sites with high and low affinity respectively, as determined by Scatchard analysis. FRTL-5/TP and FRTL-5/TA were both able to grow in TSH free medium and were nonresponsive to TSH in vitro, as tested for [3H]thymidine and [3H]uridine incorporation, iodine uptake, thyroglobulin iodination, and thyroglobulin secretion. This correlated with an approximately 100-fold decreased number of TSH binding sites compared to FRTL-5. The latter was caused by a complete absence of low affinity binding sites, whereas high affinity receptors were still detectable. The FRTL-5/TA cell line was the least differentiated one as thyroglobulin mRNA was detectable in only minute amounts and thyroid peroxidase expression could not be measured. These in vivo selected FRTL-5 cell lines offer a suitable model to investigate several aspects of TSH responsiveness, including signal transduction and postreceptor events, thyroid differentiation, and thyroid tumorigenesis.

Preferential binding of testosterone over epitestosterone by human plasma.

The main purpose of this study was to investigate if the specificity of the binding of testosterone to plasma proteins could be defined as a preferential binding of this steroid over epitestosterone. The amount of testosterone that is specifically bound was calculated using the formula: (see article) concentration, where "Ri" is the ratio (14C) testosterone: (3H) epitestosterone in plasma prior to centrifugation, "Ru" is the isotope ratio in the protein-free supernatant obtained after ultracentrifugation (149,000 x g, at 0 C, for 18 h) and "T concentration" is the testosterone concentration in plasma resulting from addition of (14C) testosterone, the endogenous steroids having been removed by preliminary charcoal extraction. The theoretical separation of the binding sites for testosterone into two populations, one non-specific with no preference for testosterone over epitestosterone, and another with absolute specificity for testosterone over its physiologically inactive stereoisomer, proved to be useful. Ovalbumin was found to be an example for non-specific, non-preferential binding. Determination of the ratio (14C) testosterone: (3H) epitestosterone in the successive fractions of various ultracentrifuged preparations showed a small but significant preference for (14C) testosterone by human and bovine serum albumin, while alpha1-acid glycoprotein had a preference for (3H) epitestosterone. Saturation curves showed at least two components: the first one, presumably corresponding to TeBG, had a higher affinity and lower capacity. This binding capacity can be accurately determined by extrapolation to the ordinate or the second component, a straight line corresponding to a binding of somewhat lower affinity and much larger capacity.

Body fat mass, body fat distribution, and plasma hormones in early puberty in females.

We examined whether there is a relationship between body fat mass or body fat distribution and hormonal profiles in the plasma of early pubertal girls. Thirty-five apparently healthy caucasian schoolgirls were selected for Tanner's breast development stage M2; they had all been classified as being stage M1 6 months earlier. Body fat mass had no relationship with the total plasma sex steroid concentration or gonadotropins. However, body fat mass was correlated with the fraction of testosterone that was not bound to sex hormone-binding globulin and considered the fraction available for biological activity. Body fat distribution, rather than body fat mass, was different in relation to the total concentrations of estrone, estradiol (E2), and testosterone as well as the percentage of available E2 or testosterone. Girls with fat localized predominantly on the hips had the highest levels of sex steroids and gonadotropins. It seems likely that this type of fat distribution is a result of ovarian activity. Girls with predominantly abdominal fat were also more obese and showed increased plasma levels of total E2 and a lower androgen/estrogen ratio in plasma, possibly due to increased aromatization, especially in abdominal adipose tissue. The findings suggest a reciprocal relationship among body fat distribution, plasma sex hormone levels, and availability of sex steroids in early female puberty.

Body fat mass, body fat distribution, and pubertal development: a longitudinal study of physical and hormonal sexual maturation of girls.

The rate at which girls progress through the stages of puberty in relation to body fat mass and body fat distribution and its relation to their hormonal profiles was studied. Sixty-eight schoolgirls participated in a longitudinal study during 3 yr. The girls were divided into subgroups with increasing skinfold thicknesses and waist-hip ratio. They were also grouped depending on Tanner's breast development classification (M2 and M3). The age at M2 was only marginally correlated with the menarcheal age, but the age at M2 and the time interval from that age to menarche was negatively correlated. Age at the onset of puberty was not related to body fat mass or distribution. The rate of pubertal development after pubertal stage M3 was negatively related to the body fat mass. Age at M2 was only correlated with estrone (E1), while the rate of pubertal development was associated with higher FSH, E1, estradiol (E2), the fraction of E2 that was not bound to sex-hormone-binding globulin (non-sex-hormone-binding globulin bound E2) and androstenedione plasma levels at the onset of puberty. Body fat distribution, rather than body fat mass was related to the total and the non-sex-hormone-binding globulin bound plasma levels of E2 and testosterone at the onset of puberty. Changes in body fat distribution in early female puberty were chiefly related to the waist circumferences. We found no evidence that body fat mass or body fat distribution triggers the onset of puberty. Body fat distribution was related to early pubertal endocrine activity. Body fat mass was negatively related to the rate of pubertal development toward menarche, but no clear indications for an endocrine-related process is found. We conclude that onset of puberty and menarche are not parallel pubertal events, and that early pubertal plasma E1, E2 and androstenedione levels are predictors for the rate of pubertal development toward menarche. We propose that the control of the onset of puberty and maturation of the hypothalamic-pituitary gonadal axis, with regard to negative feedback control, are at least partially independent. This induces on the average a "catch up" pubertal maturation in girls with a late onset of puberty.

Dietary habits, sexual maturation, and plasma hormones in pubertal girls: a longitudinal study.

The relationships between dietary components and physical or hormonal sexual maturation in 63 pubertal girls were examined. The effects of vegetable protein and dietary fiber on breast development (B = -2.0, P less than 0.05; B = -2.6, P less than 0.05, respectively) became more pronounced in a multivariate analysis, after elimination of the linear effects of body height and energy intake. From the multivariate analysis with combinations of vegetable protein, polysaccharides, and fiber in the equation, fiber appeared to be the most important factor. The gonadotropin and estradiol plasma concentrations were higher in girls who consumed less grain fiber. We conclude that a diet rich in vegetable products, especially fiber, may affect the rate of physical and hormonal sexual maturation, possibly mediated by the hypothalamus-pituitary-gonad system.

Reliability of serum prolactin measurements in women.

Prolactin, a hormone indispensable for milk secretion, has been shown to enhance the development and growth of mammary tumors in rodents; however, its importance in human breast cancer is uncertain. Serum prolactin levels are known to fluctuate considerably under normal conditions, and lack of precision in the hormone measurements may have contributed to the largely negative findings in humans to date. The purpose of this study was to investigate the reliability of prolactin measurements in women using stored serum from an ongoing prospective study of breast cancer. Separate groups of postmenopausal and premenopausal women who donated multiple blood samples at approximately 1-year intervals were studied. The reliability of a single log prolactin determination, as measured by the intraclass correlation coefficient, was 0.76 for the postmenopausal women (95% confidence interval, 0.66-0.85) and 0.48 for the premenopausal women (95% confidence interval, 0.31-0.62). These findings suggest that a single measurement is sufficient to characterize the serum prolactin level of postmenopausal women for epidemiological research. For premenopausal women, however, multiple samples are desirable. Controlling for phase of the menstrual cycle does not appear to substantially improve the reliability of premenopausal measurements.

Response of leptomeningeal metastases from breast cancer to hormonal therapy.

Intraventricular chemotherapy with radiotherapy is the standard treatment of leptomeningeal metastasis (LM) from breast cancer; this treatment increases median survival only to about 3 months and is frequently complicated by serious side effects. The authors describe two patients with LM from breast cancer who were treated with hormonal therapy, which provided a neurologic response of at least 12 months and a survival of 14+ and 19 months. Hormonal therapy can be effective and nontoxic for patients with LM from breast cancer.

Droloxifene, a new anti-oestrogen in postmenopausal advanced breast cancer: preliminary results of a double-blind dose-finding phase II trial.

Droloxifene (3-OH-tamoxifen citrate) is a novel anti-oestrogen with a higher affinity for oestrogen receptors (ER), a lower oestrogenic to anti-oestrogenic activity ratio and faster pharmacokinetics compared with tamoxifen. From May 1988 to April 1991, 369 postmenopausal women with metastatic or locally unresectable breast cancer of which the ER or progesterone receptor status was positive or unknown, were randomised to receive an oral dose of 20, 40 or 100 mg droloxifene once daily. 43 Brazilian, Canadian and European centres took part in this double-blind phase II trial. 60 women were ineligible for violation of entry criteria; 20 were inevaluable and 15 still await definitive response evaluation. 234 patients have been evaluated for response. The over-all objective response rate (complete plus partial) was 92/234 (39.3%): 23/74 (31.1%) for 20 mg, 33/74 (44.6%) for 40 mg and 36/86 (41.9%) for 100 mg (not significantly different within this dose range). There was no significant difference in time to tumour progression between the three doses. Toxicity was minimal at all doses. These preliminary results show that droloxifene is active against advanced breast cancer. Because of its outstanding preclinical characteristics, the drug warrants large-scale clinical investigation.

Thyroid function 10-18 years after mantle field irradiation for Hodgkin's disease.

Thyroid function was measured in 81 patients who had been curatively irradiated on a mantle field for Hodgkin's disease 10-18 years ago. 47 patients (58%) had elevated levels of thyroid stimulating hormone, indicating hypofunction of the thyroid gland, compared with 4.6% of controls (hospital visitors) matched for age and sex. The mean free thyroxine index (FTI) was significantly lower in patients than in controls, but all FTI values were still normal. Age at the time of irradiation, sex, time since irradiation and administration of chemotherapy were not significant factors in the development of thyroid dysfunction. A life-long awareness of the possibility of insidiously developing myxedema in these patients is strongly advocated.

Pamidronate infusions as single-agent therapy for bone metastases: a phase II trial in patients with breast cancer.

Pamidronate is a potent biphosphonate which modulates tumour-induced osteolysis (TIO) by inhibiting osteoclast-mediated bone resorption. In a phase II trial, 69 breast cancer patients with symptomatic progressive bone metastases were given infusions of pamidronate 60 mg over 1 or 4 h every 2 weeks for a maximum of 13 infusions or until progressive disease (PD) at any site. No other systemic anticancer therapy was allowed. Pain was measured using a visual analogue scale, mobility using a detailed eight-point questionnaire and analgesic intake using a six-point scale. Improvements in pain, mobility and analgesic scores occurred in 61, 50 and 30% of patients, respectively, with 33, 21 and 16% achieving a 40% improvement for > or = 8 weeks. At trial discontinuation, baseline levels of pain and mobility had improved by 27% (P = 0.001) and 20% (P = 0.004), respectively, despite a one category reduction in analgesic intake in 27% of patients. Using this relatively high dose of pamidronate, symptomatic response was independent of the number of bone metastases and also of infusion rate. The infusions were well tolerated with no major toxicities reported. Pamidronate infusions provide useful palliation for breast cancer patients with symptomatic bone metastases.

Pulmonary morbidity 10-18 years after irradiation for Hodgkin's disease.

Pulmonary function tests were performed in 78 patients who had been curatively treated for Hodgkin's disease with mantle field irradiation 10-18 years ago. Mean values of the total lung capacity (95.2%), vital capacity (VC) (95.9%), forced expiratory volume in 1 s (FEV1) (90.6%), and carbon monoxide diffusing capacity per unit alveolar volume (82.7%) showed significant deviations from the predicted normal values, standardised for age, sex, race and height. In a multiple regression analysis the normalised total dose of irradiation, the field of irradiation, and the interval since irradiation had independent negative effects on the test results. Patients reported more coughing, wheezing and dyspnoea on exertion in comparison with hospital-visitors. Their smoking habits and reported pulmonary disease were not different. It is concluded that small, but significant impairment of pulmonary function exists after a follow-up of 14 (2) years [mean (S.D.)]. The clinical impact of these findings seems, however, minimal. Further avoidance of pulmonary toxicity requires a careful quantitative study of the effects of the radiation dose and irradiated volume.

Assessment of anthracycline-related myocardial adrenergic derangement by [123I]metaiodobenzylguanidine scintigraphy.

Myocardial adrenergic neuron integrity and function were evaluated in 21 patients who had received doxorubicin or epirubicin for various malignancies. Myocardial uptake of iodine-123 metaiodobenzylguanidine ([123I]MIBG), a marker suitable for the study of myocardial neuron injury, was calculated from planar scintigraphic images after 4 h and the washout between 15 min and 4 h. In 13 patients with normal left ventricle ejection fraction (LVEF) analysed at three cumulative dose levels (no, low and middle dose), [123I]MIBG uptake tended to be significantly impaired (z = -2.772, P = 0.0056), at higher cumulative dose levels, before significant LVEF changes were observed. [123I]MIBG values were considerably decreased in 2/7 patients investigated at low cumulative dose and in 3/8 cases at the middle dose level. On follow-up, 1 of these patients, who had normal LVEF after completion of chemotherapy but whose [123I]MIBG values had progressively deteriorated during anthracycline therapy, subsequently developed congestive heart failure; another patient, whose [123I]MIBG values were impaired at the middle dose level, developed persistent reduced LVEF 5 months after completing therapy. In 8 patients, who had developed persistently, reduced LVEF at high doxorubicin cumulative dose levels, [123I]MIBG, performed in the period after chemotherapy discontinuation, was invariably abnormal. These data suggest that myocardial adrenergic derangement plays a role in anthracycline-associated cardiotoxicity: its appearance, even at low cumulative anthracycline dose levels, may reflect impairment of the intravesicular norepinephrine storage by incipient anthracycline-associated cardiac neuron injury. [123I]MIBG scintigraphy may be useful to assess myocardial adrenergic derangement during and in the follow-up of anthracycline therapy and potentially detect patients who are at risk.

Randomised trial of paclitaxel versus doxorubicin as first-line chemotherapy for advanced breast cancer: quality of life evaluation using the EORTC QLQ-C30 and the Rotterdam symptom checklist.

The aim of the study was to compare the quality of life (QL) of patients treated with single-agent paclitaxel versus doxorubicin as first-line chemotherapy for advanced breast cancer. 331 patients with advanced breast cancer were randomised, with 294 eligible for analysis. Patients completed both the EORTC QLQ-C30 questionnaire and the Rotterdam Symptom Checklist (RSCL) with six additional items, at baseline and after the third, fifth and seventh cycles of chemotherapy. A significant difference in progression-free survival in favour of doxorubicin caused a bias in the data with differences in expected completion rates of questionnaires beyond cycle three. Therefore, statistical comparisons were performed only for the first three cycles. Baseline compliance was 64% and 61% for the QLQ-C30 and RSCL questionnaires, respectively. Doxorubicin was associated with significantly more nausea/vomiting (P=0.001), loss of appetite (P=0.010) and a greater burden of disease and treatment (P=0.044), but with less bone pain (P=0.042) and rash (P=0.045) than paclitaxel. Both treatments were associated with improved emotional function and reduction in psychological distress at cycle 3. Longitudinal data suggested that doxorubicin was associated with less pain, specifically bone pain. Doxorubicin was more active but may have had more side-effects during the first three cycles. Long-term QL outcomes could not be assessed.

Efficient selection of high-affinity B cell hybridomas using antigen-coated magnetic beads.

A method for the selection of antigen-specific B cell hybridomas using antigen-coated magnetic beads is described. Stable B cell hybridoma cell lines directed against human thyroglobulin were incubated with thyroglobulin-coated beads. 2 h of incubation at 4 degrees C using bead-to-cell ratios of at least 3:1 were found to be the optimal conditions for rosette formation. Rosettes were efficiently isolated with a strong magnet. Rosette formation was antigen-specific since irrelevant hybridoma cell lines could not form rosettes, nor could BSA-coated or uncoated beads form rosettes. Free antibodies produced by the hybridoma cells were able to block rosette formation. Blocking of rosette formation permitted the identification of different and overlapping epitopes recognized by four different hybridomas. Using six stable hybridoma cell lines with different affinities for thyroglobulin, rosette formation appeared to be dependent on the affinity of the immunoglobulin membrane receptor for antigen. A correlation was observed between the affinity of the secreted antibodies and the capacity of the hybridomas to form rosettes, suggesting that this method is suitable for the selection of hybridomas producing antibodies with a high affinity for the antigen. Antigen-coated magnetic beads were found to be suitable for the efficient selection of thyroglobulin-specific hybridoma cells from bulk cultures shortly after fusion. A 300-fold enrichment of thyroglobulin-specific cells was obtained using this method.

Production of murine monoclonal antibodies against human thyroglobulin using an in vitro immunization procedure in serum-free medium.

A serum-free in vitro immunization method for the generation of hybridomas producing specific antibodies to an antigen is described. The method was tested with human thyroglobulin as antigen. The serum-free medium used (Yssel et al., 1984) consisted of Iscove's modification of Dulbecco's modified Eagle's medium, supplemented with albumin, transferrin, insulin, ethanolamine and linoleic, oleic and palmitic acids. An optimal response was obtained when splenocytes from BALB/c mice were cultured for 3 days in the presence of 1.5 nM thyroglobulin and thymocyte-conditioned medium prior to fusion with SP2/0 myeloma cells and seeding of the fused cells in microtitre plates. The frequency of positive wells, defined as the number of wells secreting anti-(thyroglobulin) antibodies/number of viable cells used for the fusion, was 1.6 X 10(-6) +/- 0.25 X 10(-6) (mean +/- SD; n = 4). Eight stable clones producing anti-(thyroglobulin) antibodies were isolated. One clone (3D12) produced antibodies reacting only with human thyroglobulin. The antibodies produced by the other clones reacted with human, murine and porcine thyroglobulins. Seven of the clones produced antibodies of the IgM class and one clone produced IgG. The specificity of 3D12 (IgM) for human thyroglobulin and the absence of any reactivity with murine thyroglobulin provides evidence for a primary response of splenocytes in culture to the presence of an antigen.

Radioimmunoscintigraphy using iodine-131-anti-CEA monoclonal antibodies and thallium-201 scintigraphy in medullary thyroid carcinoma: a case report.

This case report demonstrates the use of thallium-201 (201Tl) scans versus iodine-131- (131l) anti-CEA F(ab')2 scans in a patient with high serum CEA levels due to metastases of medullary thyroid carcinoma in the suprarenal region and sacroiliacal region. Scintigraphy using monoclonal antibodies directed against CEA showed a higher tumor uptake (0.26% dose and 0.64% dose, respectively) than a thallium scan and is believed to be promising for future radiotherapeutic applications.

Growth arrest associated changes of mRNA levels in breast cancer cells measured by semi-quantitative RT-PCR: potential early indicators of treatment response.

To find early and sensitive indicators of treatment response in breast cancer, we studied the mRNA levels of proliferation-related genes during growth arrest of the human breast cancer cell lines T47D and MCF7. A sensitive reverse transcriptase-PCR (RT-PCR) technique was used in order to monitor gene expression in small samples of cells. Estrogen-depletion and treatment with tamoxifen effectively induced a G1-arrest in both cell lines, accompanied by a decrease of the mRNA levels of histone H4, cyclin A, cyclin D1, and c-myc. Cyclin A expression decreased most strongly: up to 32-fold within 7 days. The expression of c-fos and WAF1 increased during growth arrest. In conclusion, significant changes of the levels of proliferation-related mRNAs, induced by growth arrest, can be measured in small samples of breast carcinoma cells using RT-PCR. Especially the decrease of the cyclin A mRNA level seems a potential early indicator of clinical response to tamoxifen therapy in breast cancer patients.

Diagnosis and treatment of malignant pheochromocytoma with 131I-meta-iodobenzylguanidine: a case report.

The case of a 67-year-old male patient with metastatic malignant pheochromocytoma is described to illustrate the significant improvement of the diagnosis and treatment of pheochromocytoma since the recent introduction of 131I-meta-iodobenzylguanidine (131I-MIBG). Because of the generally observed tumor heterogeneity as to 131I-MIBG uptake and radiosensitivity in advanced disease, treatment results may be improved further, if 131I-MIBG therapy is initiated when tumor load is still small.