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.

Regulation of fatty acid synthetase by progesterone in normal and tumoral human mammary glands.

Progesterone and estrogens play an important role in the control of growth, differentiation and function of mammary epithelial cells. Their mechanism of action can be studied in human metastatic breast cancer cell lines (MCF7, T47D, ZR75-1...) that contain progesterone and estrogen receptors. We used this system to try to define progestin-regulated human genes which would permit to study progestin-regulation of gene expression in cell culture and to develop clinical markers of progestin-responsiveness. This paper summarizes our investigation of the progestin-regulated 250K protein, recently identified as human fatty acid synthetase (FAS).

[Testosterone in saliva: application to the follow-up of prostate cancer]. / Testosterona en saliva: aplicación al seguimiento en el cáncer de próstata.

The basic aspects of steroid hormones transport, their tissular release and the interpretation of salivary testosterone values as a reflect of the free hormone in serum are reviewed in this article, as well as the salivary testosterone applications in several disorders, with a special emphasis on prostatic carcinoma. The usefulness of salivary testosterone in the short-term (n = 4) and long-term (n = 13) follow-up of patients affected by prostatic carcinoma after medical or surgical orchiectomy has also been studied. Our results show a correlation (r = 0.62) between salivary and serum concentration values, as well as a significative decrease (p less than 0.001) in both quantities after treatment. Both these findings and the advantages inherent to the sampling obtaining, lead to the conclusion that salivary testosterone is a good alternative to serum testosterone.

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.

[Prolactin receptors and breast cancer]. / Receptores de prolactina y cáncer de mama.

A study has been carried out on 116 mammary neoplasias in which the prolactin receptors together with other clinical and hormonal parameters have been determined. A 46.3% frequency of PRLR+ has been obtained as well as a statistically significant correlation between these and blood estradiol (p less than 0.05). PRLR- have greater survival and PRLR may act as a growth factor.

Radioiodinated ligands for the estrogen receptor: tissue distribution of 17 alpha-[125I]iodovinylestradiol derivatives in normal and tumor-bearing adult female rats.

A series of three 125I-labeled 17 alpha-iodovinylestradiol derivatives previously demonstrated to have a selectivity for estrogen receptor tissues in immature female rats were selected for further evaluation in adult female rats. In normal adult female rats, the iodovinyl analogs of moxestrol (IV beta ME2), and moxestrol-3-O methyl ether (IV beta ME2-3-OMe) demonstrated high uterine uptake (0.296-0.437 and 0.135-0.199%ID-kg/g) and selectivity (10-18:1) over the 6 h time period. Subsequent evaluation in two tumor models indicated that [125I]V beta ME2 also possessed the highest tumor uptake and selectivity in the adult female rats bearing the estrogen responsive 9,10-dimethyl-1,2-benzanthracene(DMBA)-induced mammary tumors and that this was receptor mediated. An estrogen independent tumor, the transplanted Walker 256 mammary adenocarcinoma, showed no selectivity of radioligand uptake compared with nontarget tissues. The results suggest the applicability of this agent to the in vivo detection and characterization of estrogen responsive tumors in man.

Relationship between mammographic features and hormone receptor content in patients with breast cancer.

Recurrences of breast cancer are more responsive to hormone therapy if the tumors are positive for estrogen receptors or progesterone receptors. To assess the relationship between hormone receptor content, mammographic tumor morphology, and breast parenchymal patterns, we reviewed charts and mammograms of 210 patients with primary unilateral breast cancer. Mammograms of tumors in 97 patients were divided morphologically into five groups: (1) spiculated mass, (2) architectural distortion, (3) calcifications only, (4) circumscribed mass, and (5) tumor not visible. Estrogen receptor positivity was 81% (39/48) in group 1, 37% (7/19) in group 2, 17% (2/12) in group 3, 31% (4/13) in group 4, and 60% (3/5) in group 5 (P less than .001). Mean estrogen receptor content was also significantly different among groups (P less than .001). There was no statistically significant association between tumor morphology and progesterone receptors, or between calcifications and receptor status. In all 210 patients, hormone-receptor-positive tumors showed no association with mammographic parenchymal pattern. When direct assay of estrogen receptors is unavailable, mammographic appearance of the tumor may suggest the estrogen receptor status.

Coexistence of cytoplasmic and nuclear estrogen receptors. A histochemical study on human mammary cancer and rabbit uterus.

Consecutive serial cryostat-frozen sections of 157 human mammary carcinomas and the uteri of six immature New Zealand white rabbits were stained histochemically for cytoplasmic estrogen receptor (ER) and nuclear ER by a fluorescent estrogen compound (Fluorocep Estrogen, Zeus Technologies, Inc., Raritan, NJ) and by a monoclonal antibody immunoperoxidase technique (ER-ICA, Abbott Laboratories, North Chicago, IL), respectively. The percentage of the ER-positive cells in the cancer cell population under observation was estimated and recorded. The results of the cytoplasmic ER assay were compared with those of the nuclear ER assay in each tumor; all cancers with less than 10% ER-positive cancer cells were grouped together as ER-negative tumors, the cancers with 30% or more ER-positive cancer cells as ER-positive tumors, and those with 10% to 29% ER-positive cancer cells as borderline positive. According to this manner of classification, 94% to 97% of the ER-positive mammary carcinomas diagnosed by one histochemical assay would have been identified as such by the other with no more than 10% difference in the ER-positive cell counts. The majority of ER-positive breast cancer cells and practically all of the luminal lining cells of the immature rabbit endometrium had coexistent cytoplasmic and nuclear ER. In the mammary cancers containing less than 30% ER-positive cancer cells, there was a greater (up to 20%) discrepancy in positive cell counts between the cytoplasmic ER assay and the nuclear ER assay. This discrepancy may be due to sampling errors of small clones of ER-positive cancer cells in two adjacent sections, difference in antigenic determinants between the cytoplasmic and the nuclear ER, and the binding sites in the nuclear ER being preoccupied by estrogen. The findings of this study appear to support the hypothesis that there are ER in the cytoplasm and the nucleus of the mammary carcinoma cells and the epithelial cells of the endometrium.

[A study on usefulness of the crude nuclear DHT measurement in prostatic cancer patients].

The nuclear and crude nuclear 5 alpha-dihydrotestosterone (DHT) levels were measured in patients with benign prostatic hypertrophy (BPH) and prostatic cancer (PC). In the fundamental study using BPH tissues, nuclear DHT levels were not influenced by the treatment for nuclear purification such as DTT, Triton X-100 or DNase I. The correlation between DHT levels and androgen receptor contents was found in only the crude nuclear salt extractable fractions (r = 0.748, p less than 0.01). In the relation of DHT levels to prostatic cancer patients, crude nuclear salt extractable and salt resistant DHT in both the low grade and in early stage group were significantly higher than those in the high grade and in advanced stage. Moreover, DHT levels in the both crude nuclear salt extractable and salt resistant fractions were below 5 pg/mg protein in all of clinical non-responders to endocrine therapy, and the total crude nuclear DHT, the sum of the crude extractable and salt resistant DHT, was below 10 pg/mg protein in all of the non-responders to endocrine therapy. When the relation between the total crude nuclear DHT level and the clinical course of 34 prostatic cancer patients followed for over 12 months was studied, the elevated DHT group (over 20 pg/ml protein) responded to endocrine therapy and experienced long-term remissions, but the low DHT group (below 10 pg/ml protein) did not respond to endocrine therapy. Therefore, it is suggested that the total crude nuclear DHT levels were appropriate biochemical indicators for androgen dependency in prostatic cancer patients.

A monoclonal antibody to a 48 K antigen in oestrogen-dependent human breast cancer cells.

A mouse was immunised with an antigen(s) purified by oestradiol-Sepharose affinity chromatography of pooled oestrogen-receptor positive cytosols from human breast cancer tissue. One antibody secreting clone was identified which precipitated labelled antigen and which also stained MCF-7 cells. Culture supernatant and ascites fluid were used for immunofluorescence, SDS-PAGE-Western blotting, photoaffinity labelling and binding studies. The antibody staining of MCF-7 cells was inhibited by preincubation in oestrogen-receptor positive cytosol but was unaffected by oestrogen-receptor negative cytosol. MCF-7 cells stained whether cultured in the presence or absence of oestradiol. The oestrogen-receptor negative cell lines MDA-MB-231 and MDA-MB-330 did not stain. Binding studies with 16-alpha-iodooestradiol using breast cancer tissue cytosols followed by immunoprecipitation showed activity only with oestrogen-receptor positive cytosols with optimal binding activity at 4 degrees C, unaffected by molybdate, but reduced at 25 degrees C or in the presence of 0.4 M KCl. Binding studies with MCF-7, MDA-MB-231 and MDA-MB-330 cytosols and nuclear fractions only showed activity with the MCF-7 cytosol and MCF-7 particulate fractions. The antibody recognised a 48 K species in both MCF-7 cytosol and nuclear fractions but not in the cytosol and nuclear extracts of oestrogen-receptor negative cell lines. Photoaffinity labelling using 16 alpha-iodooestradiol suggests the 48 K antigen does not bind oestradiol directly. The relationship of this antigen to the classical oestrogen-receptor and receptor complex awaits further clarification.

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.

[Estrogen receptor status and expression of carbohydrate antigens in breast cancer].

Seventy-six cases of primary cancer of the breast were studied by clinicopathological examination and immunohistological examination using monoclonal antibodies. They were composed of 40 cases positive for estrogen receptor (ER) and 36 cases negative for ER, the ER-positive rate being 52.6%. ER was detected more frequently in the postmenopausal group than in the premenopausal group, though the ER-positive rate was not significantly different between the two groups. The disease was advanced with a large tumor (greater than t3) and high-grade lymph node metastasis in ER-negative cases compared with ER-positive cases. The carbohydrate antigens specific to SPan-1, CA 19-9 and SLEX were found in 34.2%, 39.5% and 68.4% of the cases studied, respectively. These antigens were detected more often, if not significantly, in cases with a large mass and high-grade metastasis than in those with a small mass and without metastasis or with low-grade metastasis. The SPan-1-positive rate was significantly higher in ER-negative cases graded t1 or t2 than in ER-positive cases falling under the same category of tumor size. Significant differences were also noticed between the ER-negative and ER-positive groups with respect to CA 19-9-positive rate (t2 category) and SLEX-positive rate (t3 category). Similar data were obtained when our cases were classified by the degree of lymph node metastasis. In consequence, the present observations suggested that the expression of carbohydrate antigens in primary breast cancer and disease progress might be correlated with ER status.

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.

Predictors of hormone response for patients with ER-unknown breast tumours.

Characteristics of cells that are associated with the hormonal dependence of tumours are described, and it is shown that clonogenicity and hormone-induced proliferative response of breast tumours are as good markers of hormonal dependence as is oestrogen receptor. Thus tumours that formed less than 150 colonies per 500,000 cells seeded and that increased their proliferative activity 1.8-fold or more in response to hormones were the tumours that were likely to respond to endocrine treatments, whereas all other tumours were likely to be refractory to endocrine treatments. These two criteria (clonogenicity and proliferative response to growth hormones) correctly identified the response to subsequent endocrine treatments in 15 out of 17 patients with oestrogen receptor-unknown tumours. It is proposed that they may constitute a substitute for the oestrogen receptor status in patients with non-biopsiable tumours, and an additional discriminant where the oestrogen receptor assay is available.

A cytoplasmatic estrogen receptor related protein (ER-D5 Ag) in breast cancer.

A monoclonal antibody against an Estrogen Receptor Related Protein (EG-D5 AG by Amersham) was analyzed in evaluating hormone dependence in 188 breast cancers, in addition to current index of steroid receptors. The Authors observed that the concentration of this new Antigen is not related with PgR but with ER concentration. In fact, increasing the ER values, increases the concentration of ER-D5 Ag, showing a good correlation between these two tumoral markers. In regard to Progesterone Receptor ranges, the ER-D5+ves were equally distributed between PgR-ves and PgR+ves Our experience suggests the application of ER-D5 Ag as R-ves tumor screening marker and emphasizes the importance of a second level in determining therapy and prognosis in breast cancer.

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.

[Hormone independent murine carcinoma BX-1 spontaneously arising from nude mouse bearing Br-10, a hormone dependent human breast carcinoma strain].

BX-1, an adenocarcinoma spontaneously arising from nude mouse bearing Br-10, a human breast carcinoma strain was characterized. And the purification of a hormone independent murine carcinoma strain, BX-1 was found in August 1986 in three institutes where a hormone dependent transplantable human breast carcinoma cell line Br-10 has been serially passaged. The histological features of BX-1 were different from any other strains which were maintained in these three institutes. Estrogen receptor of BX-1 was negative and no estrogen dependency was observed while Br-10 was the receptor positive and the growth of Br-10 was dependent on estrogen. Although the graft of BX-1 into the thymus intact littermates was rejected, the chromosomal analysis revealed only murine chromosomes for BX-1, while both of human and murine chromosomes were detected in Br-10 tumor. By incubating Br-10 tumor in untreated female nude mice for 2 months and stimulating the growth of the tumor by exogenous estradiol, the purification of Br-10 from BX-1 could be achieved. Whereas the stability of human tumor xenografts in nude mice is confirmed, the spontaneously arising murine tumor from nude mice bearing human tumor xenografts should be considered for the experiments.