Loss of prostaglandin E receptors during progression of rat mammary tumors from hormonal dependence to autonomy.

Prostaglandin E (PGE) receptors and PGE-adenylate cyclase responsiveness were measured in tumor samples from a hormone-dependent subline of the transplantable MTW9 rat mammary tumor and from an autonomous subline derived from the hormone-dependent tumor. Scatchard analysis of the equilibrium binding data suggested that the hormone-dependent, slow-growing (MTW9A) tumors contain two major types of binding sites for PGE2: a high-affinity component (Kd less than 10(-9) M) and a low-affinity component [Kd greater than 10(-8) M]. The hormone-autonomous, fast-growing tumors (MTW9D), however, have lost more than 80% of the PG binding sites and exhibited mainly a predominant PGE lower affinity component (Kd greater than 10(-8) M). Loss of PGE receptors in autonomous tumors was not due to in vivo down-regulation of these receptors by excessive production of PGE, since both the hormone-dependent and autonomous tumors endogenously produce and release approximately the same amounts of PGE. Incubation of tumor tissues in vitro with PGE caused a significant stimulation of adenylate cyclase activity in the MTW9A tumors, whereas adenylate cyclase activity was not stimulated in the MTW9D tumors even in the presence of the nonhydrolyzable analogue of GTP, Gpp[NH]p. The results suggest that loss of PGE receptors and PGE-adenylate cyclase responsiveness occurs during progression of mammary tumors from hormonal dependence to autonomy and that the subsequent loss of cyclic AMP is associated with the uncontrolled growth characteristics of the autonomous tumors.

Estradiol receptor analysis in human breast cancer tissue by isoelectric focusing in polyacrylamide gel.

Isoelectric focusing in polyacrylamide gel combined with limited proteolysis is a simple and specific method for quantitation of estradiol receptors in breast cancer tissue. At least eight different samples can be analyzed simultaneously on one gel, and the whole procedure, including sample preparation, takes less than 7 hr. In comparison with sucrose gradient centrifugation, isoelectric focusing is more sensitive, possibly due to the short time (1.5 to 2 hr) needed for the analysis. Furthermore, only one incubation with tritium-labeled estradiol is needed for an analysis, which means that a smaller amount of tumor tissue is needed than for most other methods. This fact allows analysis of the estrogen receptor content in tumor material obtained from fine-needle biopsy.

Estrogen receptor assay in primary breast cancer and early recurrence of the disease.

Estrogen receptor assays of primary breast tumors have been related to early recurrence of the disease. A significantly longer disease-free interval was found in women whose primary tumor was estrogen receptor positive. Although there was no relationship of receptor content to stage of disease at mastectomy, the greatest difference between recurrence rates was found when the tumor had spread to the lymph nodes, especially to those in the apex of the axilla or in the internal mammary chain. Presence of estrogen receptor is closely related to histologically well-differentiated tumors, but it was found that poorly differentiated estrogen receptor-negative tumors recurred earlier than poorly differentiated receptor-positive tumors and had a very unfavorable prognosis.

Prognostic value of estrogen receptor determinations in patients with breast cancer.

The estrogen receptor content of human breast cancer specimens is related to the degree of differentiation (grade) of the tumor. In addition, patients with estrogen receptor-positive tumors experience fewer recurrences and remain disease free for a longer priod of time than do patients with receptor-negative tumors. The presence of estrogen receptor is not correlated with lymph node infiltration.

Hormone dependency of a serially transplantable human prostatic cancer (HONDA) in nude mice.

Human prostatic cancer (HONDA) serially transplanted in nude mice grew well in male mice but not at all in untreated female mice or in castrated male mice. Progressive growth in female mice was obtained by i.m. administration of 1 mg of testosterone twice a week. Estradiol inhibited the growth of the tumor in male mice to some extent; however, some growth was observed. The tumor in untreated male mice retained the histological features of poorly differentiated adenocarcinoma. Tumors in castrated male mice showed reduction in size of tumor cell nests with relative overgrowth of stroma. The tumor in androgenized female mice consisted of columnar epithelial cells with large nuclei and more abundant cytoplasms and a large glandular lumen, showing histology of moderately differentiated adenocarcinoma. High levels of human prostatic acid phosphatase (PAP) were detected in sera from untreated male mice. Testosterone markedly increased the content of serum PAP of androgenized female mice. Estradiol reduced the levels of PAP in sera from untreated male mice regardless of the tumor weight. High-affinity androgen receptors were present in cytosol and in nuclear extract of the tumor in untreated male mice. No measurable amount of progesterone or estrogen receptors was present in cytosol from untreated male mice.

Automated quantitation of immunocytochemically localized estrogen receptors in human breast cancer.

Frozen sections of breast tumor tissue have been stained using an immunoperoxidase [estrogen receptor (ER)-immunocytochemistry] kit incorporating a monoclonal antiserum [H222] to visualize nuclear human ERs. Quantitation of specific staining has been performed by manual procedures using optical microscopy and by a computer-assisted image analysis system (CAS 100). Initial investigations with a test panel of ER-immunocytochemistry-positive tumors revealed a good qualitative agreement between CAS and manual assessments. Reduced variance was, however, observed between quantified ER-immunocytochemistry results from four experienced investigators using the CAS analysis. An extended study confirmed the relationships between CAS and manual methods of assessment. These findings were evident when studies were scored either by assessment of the percentage of positively stained cells (n = 92; r = 0.919; P less than 0.01) or by H-score calculations (n = 92; r = 0.913; P less than 0.01). A good correlation was also found between CAS quantification and the results of an ER enzyme immunoassay of 48 primary breast cancer specimens (r = 0.715; P less than 0.05). In 49 cases it was possible to relate CAS-defined ER status and levels to the subsequent response of patients to endocrine therapy. ER was assessed on specimens obtained prior to commencement of treatments for recurrent breast cancer. Presuming the presence of ER to be a prerequisite for successful therapy, very good correlations between response and both status and levels of positivity were recorded. None of 16 patients with CAS-ER-negative tumors responded to treatment, while 16 of 33 (48.4%) CAS-ER-positive patients achieved an objective response according to International Union Against Cancer criteria. A relationship between response and the degree of CAS-ER positivity was obtained when the CAS score divisions of 0, 1-100, and greater than 100 (response rates, 0, 41, and 64%, respectively) were used. These data demonstrate that automated image analysis offers a reliable, reproducible procedure for quantifying ER in immunocytochemically stained sections. It has potential advantages over manual procedures, providing less opportunity for subjective influences in scoring sections. Future advances in software design should further reduce elements of subjectivity and increase both the speed and reliability of results. We anticipate image analysis becoming a valuable tool in investigations concerning, for example, the influence of heterogeneity of steroid receptor distribution on the rate of recurrence of breast cancer after mastectomy and in the clinical course of the disease.

Correlation between clinical response to hormone therapy and steroid receptor content in prostatic cancer.

Hormonal therapy is the dominating form of treatment for prostatic carcinoma. The majority of cases (80%) are well controlled for varying times with this regimen. However, thus far there have been no adequate methods to predict in which cases hormonal therapy is of less benefit. Measurement of cancer tissue content of intracellular hormone receptors constitutes progress toward a more individualized therapy in prostatic carcinoma. In this study biopsies from 16 cancer patients were taken before therapy was given, and the specimens were analyzed with regard to content of specific methyltrienolone-binding sites. A correlation has been made between receptor content and clinical response to hormonal therapy in each case. Twelve specimens contained measurable amounts of steroid receptors. Of these, one patient died during irradiation therapy before onset of hormonal treatment. However, of the remaining 11 patients, 9 responded well to hormones (9/11 approximately 82%). The two receptor-positive nonresponders had the lowest measurable receptor levels in the series. Four specimens contained no detectable amounts of receptors. Three of these patients showed no response to therapy (3/4 = 75%) but one was "false negative." Our data indicate that steroid receptor analysis may become a valuable diagnostic tool in individualizing the therapy for prostatic cancer.

Heterogeneity in the hormonal responsiveness of clones derived from the 13762NF rat mammary tumor.

The transplantable hormone-responsive rat mammary adenocarcinoma 13762NF was dissociated with collagenase and hyaluronidase. Cells were cloned directly or lines were established from mass cultures and cells from these lines were cloned. Clones differed in cellular morphology, colony morphology on plastic or in collagen gel, growth rate, growth response to hormones, and hormone receptor levels. Growth response to prolactin, estradiol, progesterone, cortisol, and epidermal growth factor (EGF) was determined by culturing the cells within collagen gel and using a serum-free medium base of DME/F12 (1:1) with insulin, linoleic acid, and BSA. The clones varied in their hormone responses, with all 20 of the clones tested responding to cortisol in combination with EGF. Some clones would respond to EGF, cortisol, or progesterone when used alone. None of the clones tested could be stimulated by prolactin or estradiol. Receptor levels for estradiol, progesterone, glucocorticoids, and EGF were assessed in 3 selected clones differing in their hormone responsiveness. Receptor levels appeared to correlate with hormonal sensitivity. Selected clones transplanted into female F344 rats produced carcinomas with histopathologies similar to the original tumor.

Stimulative effect of physiological doses of androgen or pharmacological doses of estrogen on growth of Shionogi carcinoma 115 in mice.

It was generally accepted for 20 yr that the growth of Shionogi carcinoma 115 (SC115) is stimulated only by androgen. In the present study, the growth-stimulative effect of estrogen alone on SC115 tumors was examined in castrated mice. Daily injections of physiological doses of 17 beta-estradiol did not enhance the tumor growth. However, high doses of 17 beta-estradiol (10-100 micrograms/mouse/day) significantly stimulated the growth of tumors in a dose-dependent manner. Since high doses of diethylstilbestrol (10-50 micrograms/mouse/day), which does not bind to androgen receptor, could markedly stimulate the growth of tumors and since antiandrogen (cyproterone acetate) failed to inhibit the growth stimulation induced by high doses of 17 beta-estradiol, it is concluded that high doses of 17 beta-estradiol, which binds to androgen receptor with relatively low but significant affinity, enhance the tumor growth not via the androgen receptor system. The growth speed, histological type, content, and affinity of androgen, estrogen, and progesterone receptors and pattern of newly synthesized proteins labeled in vitro with [35S]methionine of tumors grown by high doses of estrogen were not significantly different from those of the original SC115 tumors grown in normal males. Furthermore, seed tumors from one to six generations grown by pharmacological doses of estrogen alone could rapidly grow only in normal males and not in castrated males. The present findings demonstrate that the growth of SC115 tumors in vivo is stimulated by physiological doses of androgen or pharmacological doses of estrogen.

Expression of the gene encoding a prolactin-inducible protein by human breast cancers in vivo: correlation with steroid receptor status.

We have previously reported the identification and characterization of a prolactin-inducible protein (PIP) as well as the cloning of the gene encoding PIP in cultured human breast cancer cells. We now present three lines of evidence that the gene encoding PIP is also expressed by some human breast cancers in vivo: detection of PIP immunoreactivity in the serum of some breast cancer patients; immunohistochemical detection of PIP in breast cancer sections; and the presence of PIP mRNA, detected by complementary DNA hybridization in human breast biopsy samples. In a preliminary study using Western blot analysis authentic PIP was detected in the serum of some patients with breast cancer. Subsequently the sera of 234 unselected patients with breast cancer were assayed for the presence of PIP using a specific radioimmunoassay. Thirty-five % of these sera contained detectable PIP (i.e., greater than 3 ng/ml). As well as were able to show by immunohistochemical techniques that PIP immunoreactivity was present in some human breast biopsy specimens. Levels of estrogen receptor, progesterone receptor, and PIP mRNA were determined in an unselected population of 51 human breast tumor biopsies. Sixty-one % of these tumors had detectable PIP mRNA; a positive correlation (r = 0.52; P less than 0.01) was found between PIP mRNA levels in breast biopsy samples and estrogen receptor content, a known prognostic indicator in human breast cancer.

Influence of pituitary grafts or prolactin administrations on the hormone sensitivity of ovarian hormone-independent mouse mammary MXT tumors.

The sensitivity of MXT mouse mammary tumors to ovarian hormones was assessed by the following parameters: growth in vivo; presence or absence of estrogen receptors and progesterone receptors; and histological differentiation. A spontaneous evolution from hormone sensitivity (HS) to hormone independence (HI) was observed when the tumors underwent monthly transplantation onto intact recipient mice, with the tumors fulfilling the criteria of HI tumors after the 12th transplantation. In contrast, the tumors recovered most of the criteria of hormone sensitivity when pituitary isografts were placed under the kidney capsules of HI tumor-bearing animals or when these animals received daily administrations of prolactin over several months. Sensitivity to 17 beta-estradiol, progesterone, or prolactin was further assessed by actinomycin binding on the nucleus and thymidine labeling index, both measured by autoradiography. These technical approaches revealed that 17 beta-estradiol and prolactin stimulated the thymidine labeling index of both HI (despite the lack of detectable estrogen receptors) and HS MXT tumors whereas progesterone influenced only that of HS cancers. The three hormones significantly stimulated [3H]actinomycin D binding within HS tumors but not within HI ones. However, such "HI" tumors were characterized by increased actinomycin binding and thymidine labeling index in comparison with HS neoplasms. Thus, all the data presently reported strongly suggest that prolactin is able to restore the hormone-sensitive phenotype within so-called MXT hormone-independent tumors.

7,12-Dimethylbenzanthracene induced mammary tumours in Wistar rats by 'air pouch' technique--a new approach.

An 'air pouch' technique of inducing highly localized, transplantable, estrogen-dependent adenocarcinomas of the mammary gland in Wistar rats with 7,12-dimethylbenzanthracene (DMBA) is described. The induction time was 67-80 days compared to 150-180 days by the conventional technique. Using this model estrogen receptor status, transplantation and effect of exogenous hormones on tumour growth have been studied. It is felt that this model might be a useful one for studying the biochemical mechanisms involved in hormone-dependent experimental mammary cancers.

Proteinase-like peptidase activities and oestrogen receptor levels in breast cancer tissue.

The relationship between proteinase-like peptidase activities and oestrogen receptor levels and status in breast cancer tissue homogenates from 61 patients with breast cancer has been evaluated. With Spearman's rank-order correlation analysis, significant positive correlations were observed between receptor levels and the activities of cathepsin-(B + L)-like, cathepsin-H-like, trypsin-like, plasminogen-activator-like and elastase-like peptidases. In addition, the activities of all but the latter enzyme were significantly higher in patients with receptor-rich tumours than in receptor-poor tumours, and this may have implications for future treatment regimens for patients with oestrogen-receptor-rich tumours. The findings reported are consistent with the suggestion that in breast cancer there may be an association between steroid receptors and proteolytic enzymes such that the release of these enzymes may be under hormonal control.

Cellular localization of estrogen binding sites in human breast cancer.

Biopsy specimens from 52 consecutive cases of primary human breast cancer were collected over a period of seven months and included in a study for histochemical localization of estrogen binding sites (EBS), using a fluorescein labelled Estradiol conjugate. Cryostat frozen sections from each tumor were examined to determine the localization of the tracer and to evaluate the percentage of positive cells in a given tumor. Results are correlated with the values of the biochemical assay for the estrogen receptor (ER) protein done on the same tumor. The localization of EBS by a tracer is a simple technique that can be done and interpreted in any surgical pathology laboratory. It was concluded that this method could be a valuable supplementary technique to the biochemical assay; allowing more accurate selection of patients and prediction of their response to endocrine therapy. Clinical follow-ups are extremely necessary to evaluate the suitability and the accuracy of the technique in choosing breast cancer patients for endocrine manipulation.

Uveal melanomas presenting during pregnancy and the investigation of oestrogen receptors in melanomas.

We observed a young woman who showed growth of a choroidal melanoma over the course of 2 pregnancies, with subsequent enucleation of the eye. This is the first such documented case. In addition 4 other women with uveal malignant melanomas presented during pregnancy. This observed number of pregnancies (5) was greater than the expected number (2.1) among women of childbearing age who underwent enucleation with subsequent analysis in our pathology laboratory. However, this difference was not statistically significant. Further, more females 44 years of age or younger underwent enucleation for malignant melanoma than men of comparable age. Evaluation of these cases led us to propose that there is a subset of patients whose ocular melanomas are hormonally responsive. We therefore analysed uveal melanoma and choroidal tissue from 7 patients, including one of the pregnant women, for the presence of oestrogen receptors. No specific oestrogen binding was found. The possibilities that other hormones are involved or an immunological mechanism is operative are discussed.

Sex hormone receptors in intracranial tumours and normal brain.

Fifty-four intracranial neoplasms (29 meningiomas, 16 gliomas, seven acoustic neuromas and two cerebral metastases) and nine specimens of normal brain were evaluated for specific progesterone and oestrogen receptor proteins using a dextran-coated charcoal assay and Scatchard plot analysis. Sixteen meningiomas (55%) were progesterone receptor (PgR) positive (median 52; mean 120; range 10-486 fmol mg-1 cytosol protein), whilst all were oestrogen receptor negative (ER-). Two (28%) of the acoustic neuromas contained small amounts of PgR protein, but all seven were ER-. None of the gliomas, cerebral metastases or specimens of normal brain contained ER or PgR protein. Analysis of PgR status and clinicopathological data suggest that there is no predictive correlation between PgR status and the patients age, sex, reproductive status, tumour histology or tumour behaviour. These results again suggest that in meningiomas PgR proteins are not modulated by oestrogens acting through ER. This finding may explain the failure of antioestrogen therapy to influence the growth of meningiomas. The significance of PgR protein in intracranial meningiomas is discussed with respect to tumour heterogeneity and implications for research with gene probes.

The assessment of disease aggressivity in stage D2 prostate cancer patients (review).

Suppression of androgen levels in blood of stage D2 prostate cancer patients has been the prominent treatment for advanced prostate cancer. However, the duration of hormone sensitivity of prostate tumor is variable. The type of initial response to hormonal treatment, the length of response and patient's survival are in direct association with disease aggressiveness. Recently, an arithmetic formula expressing disease aggressivity was computed using pretreatment values of prostatic acid phosphatase (P.A.P.), alkaline phosphatase (A.P.), degree of tumor differentiation and number of bone metastases. This aggressiveness score was related to disease response and patients outcome receiving hormonal treatments. The use of an arithmetic formula to express disease aggressivity could result in a subdivision of the disease. The identification of the subgroup of stage D2 patients destined not to benefit from hormonal manipulation could change the strategies employed up until today for the treatment of advanced prostate cancer.

Correlation of estrogen, progesterone, and androgen receptors in breast cancer.

Breast cancer was induced in female Holtzman rats by intragastric administration of 7,12-dimethylbenz[a]antracene (DMBA). At tumor maturity, biopsies of viable tissue were obtained, frozen, and then assayed for estrogen, progesterone, and androgen receptor content. By simple linear regression analysis, progesterone receptor levels significantly correlated with both estrogen and androgen receptor levels, whereas estrogen and androgen receptor levels did not correlate with each other. Multiple regression analyses further substantiated the predictive value of the progesterone receptor for the other two hormone receptors. Knowledge of breast tumor androgen receptor levels may further enhance the value of the estrogen receptor and progesterone receptor in hormonal responsiveness. Further, the progesterone receptor may be the most sensitive of the steroid hormone receptors for selecting patients likely to respond to hormonal therapy.

Characterization of prolactin receptors and their distribution among American and Israeli women with breast cancer: implications for prediction of hormonal dependency and treatment.

A micro-method is reported for the determination and characterization of prolactin (PRL) receptors in human breast cancer specimens using either intact biopsy tissue or the pellet fraction remaining from biopsies previously processed for steroid hormone receptors. Labeled human PRL is used as the ligand. The specific PRL-binding of 307 human breast cancer specimens was evaluated by this micro-method. A significant level of specific PRL-binding (3-25 fmol per mg membrane protein) was detected in 41% of US breast cancer patients and 42% of Israeli patients. Scatchard analyses, performed on pooled membrane fractions with the highest specific PRL-binding revealed one class of receptors having a dissociation constant, Kd = 4.1 x 10(-9), and specific binding capacity, Bmax = 1.3 pmol per mg protein. These preparations were exposed to 3 M MgCl2, which dissociates the endogenous-bound PRL from the hormone-receptor complex and allows the characterization of the "total" PRL receptors. Two classes of receptors were then revealed. One class of receptors showed high affinity (Kd1 = 8.1 x 10(-10) M) and low capacity (Bmax1 = 335 fmol per mg protein), while the other possessed lower affinity (Kd2 = 8.2 x 10(-8) M), but a higher capacity (Bmax2 = 34.4 pmol per mg protein). Since PRL facilitates the growth of human breast cancer cell lines, these results indicate that a high proportion of human breast cancers may be PRL dependent. Routine determination of PRL receptors in biopsies of human breast cancer may permit the selection of a better treatment, since it is possible that the reduction in PRL levels could inhibit those tumors which are prolactin dependent.

A new assay to assess steroid-hormone responsiveness in head and neck cancer.

A new assay has been developed to predict the effectiveness of steroid-hormone therapy in various tumors. The Biopsy Nuclear Binding assay measures the amount of biologically active receptor that binds both steroid hormone and acceptor sites in the nucleus. This assay does not measure the entire receptor population, only those that are biologically active; therefore, it should more accurately predict the response to steroid-hormone therapy. We applied this assay effectively in breast and endometrial carcinoma. Preliminary studies have shown that 40% of patients with head and neck squamous-cell carcinomas have biologically active (nuclear bound) progesterone receptors. If nuclear binding predicts a remission with hormonal therapy, then the quality of life of appropriately selected patients could greatly improve.