Dextran-coated Charcoal Method Research Articles

Androgen-binding components of human semen.

Androgen-binding components were detected in human semen, using the dextran-coated charcoal method. Pre- and postvasectomy semen samples did not show significant differences in ability to bind dihydrotestosterone. Electrophoresis conducted in polyacrylamide gels in which tritiated dihydrotestosterone had been incorporated disclosed the presence of androgen-binding protein with properties characteristic of the ABP of Sertoli cell origin in ram semen but ABP was not detectable in human semen, even after concentrating it two or four fold. The binding of dihydrotestosterone was slightly greater in the later portion of split-ejaculate samples, but binding was found in the earlier fraction and in prostatic fluid, suggesting that androgen-binding components enter human semen with secretions of the prostate and seminal vesicle glands. It was concluded that the extent of androgen-binding by human seminal fluid is not a measure of Sertoli-cell function.

Estrogen receptor in breast cancer of the Japanese.

The estrogen receptor was assayed, using the 2,000g supernatant and dextran-coated charcoal method, in 243 tissue samples from human breast cancer, benign breast diseases, macroscopically normal breast tissues, normal uterine myometrium, and uterine myoma. The estrogen receptor was found to be positive in 52.1% of 98 primary breast cancer and in 54.1% of 24 metastatic tumors. The receptor in the breast cancer was found to be similar to that in normal uterine myometrium in the binding character; that is, the dissociation constant of 10(-9) approximately 10(-11) M and number of binding sites of 0 approximately 2,800 fmol/mg protein. There was no correlation between the presence of the receptor and some clinical factors such as menopausal status, age of the patient, urinary 17-ketosteroid excretion, clinical stage of cancer, tumor size, positive or negative axillary lymph node metastasis, histological type, metastatic site of the cancer, or disease-free interval. The estrogen receptor appeared to be retained by metastasis of cancer, and this may lead to the use of the receptor assay with mastectomy specimens for the prediction of response to hormonal therapy in future recurrence of malignancy. Furthermore, it may be possible by this assay to select patients suitable for adjuvant therapy with hormones at the time of mastectomy. A good correlation was found between the presence of the receptor and response to the major endocrine ablation therapy in patients with advanced or metastatic breast cancer. When the receptor was negative in the cancer tissue, the change of response to the endocrine therapy was minimum. On the other hand, if the cancer contained the receptor, approximately 60% of the patients with metastatic or advanced breast cancer responded well to the major endocrine ablation therapy. Thus, the estrogen receptor of breast cancer in Japanese patients appears to bear a close resemblance to that reported in Western patients in its incidence and the correlation to some biological characteristics of the cancer.

Prognostic relevance of androgen receptor detection in operable breast cancer

Androgen receptor (AR) is relevant for prognostication in breast cancer. Different determination methods and cut-off levels hamper interpretation and comparisons of studies. Long-term prognostic evaluation of different AR assays in patients comprising operable breast cancers is scarce. AR was evaluated in 120 primary tumors using the dextran-coated charcoal method (charc-AR), and quantitative immunohistochemistry (IHC) on whole sections (WS) and tissue microarrays (TMA). Nuclear and cytoplasmic-AR localization was determined, and the prognostic importance of AR assays was assessed. Comparisons and correlations with the mitotic activity index (MAI), estrogen receptor (ERalpha), progesterone receptor (PR), HER-2, and histological grade (WHO I-III) were made. Nuclear-AR in WS, but not charc-AR, strongly correlated with MAI (P = 0.001). However, prognostic information appeared in univariate survival analyses only. Nuclear-AR in TMA was not prognostic. Charc-AR was independent prognostic in node positives both for relapse free survival (RFS) and breast cancer specific survival (BCSS). Both charc-AR and IHC cytoplasmic-AR provided independent prognostic survival information for BCSS in women <55 years. Methods that can detect AR localized in the cytoplasm yield important prognostic information, and further studies in patients with operable breast cancer are warranted.

Androgen Receptor Determination in Breast Cancer

Androgen receptor (AR) expression is prognostic for breast cancer. Techniques to determine AR levels include the dextran-coated charcoal method (charc-AR), which measures AR levels in both normal and cancer cells; immunohistochemical analysis (1HC-AR) of whole sections (WS), which targets cancer cells but relies on subjective assay interpretation; and IHC of tissue microarray (TMA) samples, an approach that can be inaccurate when AR heterogeneity is present. This study compared charc-AR (n=151) with quantitative IHC analysis (n=138) of WS and TMA samples (1.7 mm cylinder samples; 2.3 mm). Charc-AR results correlated poorly with IHC-WS and IHC-TMA results due to the presence of AR-positive noncancer cells. IHC-WS revealed intertumor and intratumor AR heterogeneity. Consequently, the IHC-WS and IHC-TMA results were similar for tumors in which the percentage of AR-positive WS samples was >80% (31% of tumors), but differed in other tumors due to TMA false negatives (33%). Computer simulation was used to determine the optimal number and size of TMA samples for reliable results, and showed that five to eight 1.7 mm samples, or up to sixty-four 0.6 mm samples, were needed for reliable AR determination for most tumors. Thus, AR determination by charc-AR is inaccurate due to evaluation of both normal and cancer cells. IHC is more sensitive and specific than charc-AR in WS samples, but not when applied to single or a few TMA samples.

Role of Glucocorticoid Receptor in Serosa-Involved Gastric Carcinoma After Gastrectomy

Glucocorticoid receptor (GR) was first found in the cytosol of gastric cancer tissue more than 15 years ago. At present, most gastric cancers are diagnosed at the advanced stage. To elucidate the role of GR in gastric cancer, the GR levels of the cancer tissue of 75 consecutive patients with grossly serosa-involved gastric carcinoma were determined by the dextran-coated charcoal method. The clinicopathologic characteristics and long-term survival duration were compared in patients with GR-positive and GR-negative cancer cells. We found that GR could be detected in the cytosol of cancer cells in 31 (41.3%) of the gastric cancer patients with a median concentration of 18.5 (range, 1.03-73.9) fmol/mg protein. No significant differences could be found in any clinicopathologic characteristic between the patients with GR-positive and GR-negative cancers. After multivariate analysis, gross Borrmann's type, metastatic lymph node number, and GR positivity were the independent prognostic factors after gastrectomy for serosa-involved gastric carcinoma. GR-positive gastric cancer had a worse survival rate than GR-negative gastric cancer. Multimodality adjuvant therapies should be considered in patients with GR-positive serosa-involved gastric carcinoma.

Estradiol/Androgen Receptors During Aging: Microsomal Distribution in Human Benign Prostatic Hypertrophy

Estradiol and androgen receptor levels were determined in benign prostatic hypertrophy tissue by means of dextran-coated charcoal method. 80% of the cases contained estrogen receptor, whereas 85% of cases contained the androgen receptor. In 50 year old patients, nuclear estradiol and androgen receptors were 48% and 34% higher than the cytosolic values, respectively, whereas in 70 year old patients, the levels of nuclear estradiol and androgen receptors were 6% and 57% higher than the cytosolic values, respectively. Nuclear androgen receptor in 50 year old patients was 34% higher than the estradiol receptor, and in 70 year old patients, androgen receptor was 23% higher than the estradiol receptor. Cytosolic values of estrogen receptor in 50 year old patients were diminished 50%, whereas in 70 year old patients, the ER value was increased 40% with regard to androgen receptor. On the other hand, estradiol receptor values measured in microsomal fractions, and were higher when compared to androgen receptor lev...

Estrogen Receptor Testing of Breast Cancer in Current Clinical Practice: What's the Question?

Estrogen Receptor Testing of Breast Cancer in Current Clinical Practice: What's the Question?

Epidermal growth factor receptors on human breast cancers.

Epidermal growth factor (EGF) is a potent mitogen for many tissues including breast. The receptor for EGF (EGF-r) has the same peptide sequence as a known oncogene. Sixty-one human breast cancers and nine associated lymph node metastases have been examined for the presence of EGF receptors by competitive binding and immunocytochemical techniques. Oestrogen receptor analysis on the same specimens was performed by the dextran coated charcoal method and compared with EGF-r status. These were correlated with the clinical findings and histological data. An inverse relationship between EGF-r and ER was found (chi 2= 7.81, P less than 0.01) with a higher incidence of EGF-r positive tumours in the metastatic group (chi 2 = 14.51, P less than 0.001). EGF-r positive tumours were of poor differentiation and had characteristics associated with poor prognosis.

A study of cytoplasmic oestrogen receptors in patients with breast cancer in a district general hospital.

The tumors of 61 patients suffering from primary adenocarcinoma of the breast were assayed for oestrogen receptors (ER), using the dextran-coated charcoal method. A statistically significant relationship (P less than 0.05) was found to exist between ER status and the histological grade of the tumour. No relationship was found between ER status and either tumour size or the pathological state of the axillary lymph nodes. It is concluded that it is a practical proposition to establish an ER assay service in a district general hospital, with subsequent benefits for the individual patient.

PROSTATIC CANCER/BENIGN PROSTATIC HYPERTROPHY. SUBCELLULAR DISTRIBUTION OF ESTRADIOL/ANDROGEN RECEPTORS

Six microsomal population of estradiol and androgen receptors have been characterized in human benign prostatic hypertrophy (BPH) and prostate cancer (PCa). Estradiol receptor (ER) and androgen receptors (AR) were extracted using 0.6 M KCL and determined by the dextran-coated charcoal method. ER and AR levels were smaller in BPH plasma membranes (PM) than in Pca cases. For functions 3, 4, 6, the ER values in PCa were 25-38% less with regard to BPH ER values. Whereas in PCa, AR values obtained in all fractions were higher when compared to BPH AR values. In benign prostatic hypertrophy and prostatic cancer, ER and AR levels were significantly higher in the nuclear fraction. In the nuclear fraction, ER and AR levels in BPH and PCa were significantly different. The subcellular distribution of AR and ER in BPH and PCa constitutes a reservation mechanism and processing a receptors for their continued growth.

Estradiol/Androgen Receptors During Aging: Microsomal Distribution In Human Benign Prostatic Hypertrophy

Estradiol and androgen receptor levels were determined in benign prostatic hypertrophy tissue by means of dextran-coated charcoal method. 80% of the cases contained estrogen receptor, whereas 85% of cases contained the androgen receptor. In 50 year old patients, nuclear estradiol and androgen receptors were 48% and 34% higher than the cytosolic values, respectively, whereas in 70 year old patients, the levels of nuclear estradiol and androgen receptors were 6% and 57% higher than the cytosolic values, respectively. Nuclear androgen receptor in 50 year old patients was 34% higher than the estradiol receptor, and in 70 year old patients, androgen receptor was 23% higher than the estradiol receptor. Cytosolic values of estrogen receptor in 50 year old patients were diminished 50%, whereas in 70 year old patients, the ER value was increased 40% with regard to androgen receptor. On the other hand, estradiol receptor values measured in microsomal fractions, and were higher when compared to androgen receptor levels (mean percentage +/- 50%). These findings suggest that the biological effects due to the subcellular distribution of AR or ER and that their microsomal distribution would constitute a reserve mechanism and activation of receptors for the continued growth of benign prostatic hypertrophy.

MUC3 and MCA serum levels and steroid receptor content in breast cancer.

Mucins are an important class of complex glycoproteins expressed by many epithelial cells and their malignant counterparts. The aim of this study was to determine the serum levels of MUC3 and mucin-like carcinoma-associated antigen (MCA) in patients with primary breast cancer and to analyze the possible relationships between these two mucins and the steroid receptor status. The preoperative basal serum levels of MUC3 (ELISA assay with monoclonal antibody 1143/B7) and MCA (EIA assay with anti-MCA mouse monoclonal antibody b-12) were determined in 44 patients with breast cancer while estrogen receptor (ER) and progesterone receptor (PgR) levels were measured by the dextran-coated charcoal method in the cytosol of neoplastic tissue. MUC3 was expressed in 43/44 serum samples while high MCA serum levels were found in 16/44 only; the mean values of both markers did not correlate with menopausal status, tumor size, nodal involvement or ER. The only significant difference observed was a lower median value of MCA in patients with small tumors (T1-T2). No statistically significant correlation between MUC3 and MCA, MUC3 and ER or MCA and ER was observed; a statistically significant direct correlation between MUC3 and PgR+ status and a statistically significant inverse correlation between MCA and PgR+ were observed. Our results suggest that further investigations are necessary to establish whether progesterone can modulate MUC3 and MCA expression in breast cancer.

Effects of tamoxifen on human squamous cell carcinoma lines of the head and neck.

Tamoxifen (TAM) is a well-tolerated compound in the treatment of breast cancer and is primarily considered to act by competition with estrogen receptors (ER). Here we investigated the in vitro efficacy and potentially underlying mechanisms of TAM in established cell lines of squamous cell carcinomas of the head and neck (SCCHN). Using proliferation and apoptosis assays the antitumor activity of TAM in five SCCHN and the breast carcinoma line MCF-7 (positive control) was determined. MCF-7 was more sensitive to low-dose TAM (below 1 microM), whereas SCCHN showed significant growth inhibition at higher TAM concentrations (5-10 microM). Growth curve analysis and apoptosis assays were indicative for a cytostatic effect of low-dose TAM and high-dose TAM led to cell loss by apoptosis in sensitive SCCHN. In order to further characterize the observed antitumor effects we determined the amount of steroid hormone receptors with the dextran-coated charcoal method and immunocytochemistry. In addition, production of transforming growth factor (TGF-)-alpha, -beta1 and -beta2 was measured by ELISA, and protein kinase C (PKC) activity was assessed with a radioligand assay. Except MCF-7, none of the SCCHN lines was positive for ER. TAM caused decreased TGF-alpha and increased TGF-beta levels in MCF-7, but not in SCCHN supernatants. Furthermore, the antiestrogen reduced PKC activity in MCF-7, but not in SCCHN. In the present in vitro system, the observed antitumor activity of high-dose TAM in SCCHN cannot be explained by estrogen antagonism, alterations of TGF-alpha/beta levels or decreased PKC activity.

Telomerase activity in breast cancer in Western India (Gujarat).

The purpose of this study was to examine the association of telomerase activity with clinical and histopathological prognostic variables in primary breast cancer (n=64). Telomerase activity in breast cancer was also compared with that in benign (n=10) and non-malignant tissues (n=8; post-lumpectomy tissues histopathologically defined as containing no residual tumor). The parameter was assessed using the Telomerase PCR ELISA kit. Values above OD 0.120 were considered positive. Estrogen and progesterone receptors (ER and PgR) were assayed by the dextran-coated charcoal method and levels >10 fmol/mg cytosol protein were taken as positive. Telomerase activity was detected in 20% and 50% of the patients with benign lesions and primary breast cancer, respectively, and in 50% of post-lumpectomy breast tissues histopathologically defined as containing no residual tumor. Telomerase activity was present in all stages of breast carcinoma and showed a significant inverse correlation with lymph node status (p=0.006), lymphatic invasion (p=0.035) and necrosis (p=0.033). Moreover, when stage II patients were grouped according to nodal involvement, a trend towards significance was observed (p=0.055). No correlation was observed with ER and PgR. The results of our study suggest that telomerase activity might be associated with the presence of cancer cells. Furthermore, telomerase activation may occur early in breast cancer and may be periodically downregulated during subsequent tumor progression.

Telomerase activity in breast cancer in Western India (Gujarat)

The purpose of this study was to examine the association of telomerase activity with clinical and histopathological prognostic variables in primary breast cancer (n=64). Telomerase activity in breast cancer was also compared with that in benign (n=10) and non-malignant tissues (n=8; post-lumpectomy tissues histopathologically defined as containing no residual tumor). The parameter was assessed using the Telomerase PCR ELISA kit. Values above OD 0.120 were considered positive. Estrogen and progesterone receptors (ER and PgR) were assayed by the dextran-coated charcoal method and levels >10 fmol/mg cytosol protein were taken as positive. Telomerase activity was detected in 20% and 50% of the patients with benign lesions and primary breast cancer, respectively, and in 50% of post-lumpectomy breast tissues histopathologically defined as containing no residual tumor. Telomerase activity was present in all stages of breast carcinoma and showed a significant inverse correlation with lymph node status (p=0.006), lymphatic invasion (p=0.035) and necrosis (p=0.033). Moreover, when stage II patients were grouped according to nodal involvement, a trend towards significance was observed (p=0.055). No correlation was observed with ER and PgR. The results of our study suggest that telomerase activity might be associated with the presence of cancer cells. Furthermore, telomerase activation may occur early in breast cancer and may be periodically downregulated during subsequent tumor progression.

Roles of the glucocorticoid receptor in resectable hepatocellular carcinoma

Background. The glucocorticoid receptor (GR) was discovered in the cytosol of hepatocellular carcinoma (HCC) cells more than 10 years ago. However, the influence of the GR on the prognosis of HCC after liver resection remains unclear. Methods. Ninety-two consecutive patients with HCC who survived liver resection and who did not receive any preoperative neoadjuvant therapy were enrolled in this study. The GR level in cytosol of cancerous tissue was determined by the dextran-coated charcoal method. The clinicopathologic characteristics and long-term prognosis of patients with GR-positive tumors (GR-positive group) were compared with those of patients with GR-negative tumors (GR-negative group). Results. GR was found in 63 patients (68.5%) with a mean ± SEM concentration of 26.97 ± 4.05 fmol/g protein. There were no significant differences in patient clinicopathologic characteristics between GR-positive and GR-negative groups. The 5-year disease-free and actuarial survival rates for GR-positive and GR-negative groups were 21.6% and 44.4% (P =.002) and 57.2% and 83.3% (P =.0003), respectively. After multivariate analysis was performed, GR positivity was found to be an independent prognostic factor of disease-free and actuarial survival after liver resection for HCC. Conclusions. The GR can be found in the cytosol of most HCCs and is an independent prognostic factor of HCC after liver resection. Patients with GR-positive HCC have lower survival rates than those with GR-negative HCC. (Surgery 2002;131:19-25.)

Immunohistochemical assessment of oestrogen and progesterone receptors: correlations with the DCC method and clinical outcome in primary breast cancer patients

Two different methods to determine steroid receptors were analysed with respect to their ability to estimate prognosis in primary breast cancer patients. The immunohistochemical assay (IHA) was compared with the dextran-coated charcoal (DCC) method of receptor determination. A random sample of 281 patients with invasive ductal carcinoma was drawn from 841 consecutive patients with primary breast carcinoma treated at Odense University Hospital between 1 January 1980 and 31 December 1990. Receptor determination by the DCC method had been carried out previously in 164 patients for the oestrogen receptor and in 132 patients for the progesterone receptor. The former group was reassessed by IHA with the antibody ER1D5, and the latter with the antibody PgR-ICA. The median follow-up time was 8.3 years (range 2.9–12.9 years). A cutoff of zero was used for the DCC method. Immunohistochemical results were quantified by counting in systematically random sampled fields of vision and values above zero were considered to be positive. Overall agreement of positive and negative cases was 86% for the oestrogen receptor and 83% for the progesterone receptor. Although the study included a limited number of patients, receptor positive cases fared better than negative cases in all situations. Investigation of the prognostic power revealed that classification based on IHA allowed better discrimination of patients than classification based on the DCC method. The reason for this difference might be because distinction between benign and malignant tissue is possible using the IHAmethod. Thus, IHAresults appear to be more clinically relevant.

Usefulness of Estrogen Receptor Detection Using Archival Papanicolaou-Stained Smears

To examine estrogen receptor (ER) detection using cytologic specimens and to compare the results with those obtained by the dextran-coated charcoal (DCC) method and enzyme immunoassay (EIA). Immunocytochemical staining was conducted on 60 cases of breast cancer resected at our hospital between April 1993 and November 1997 in which ER had been measured by DCC or EIA. Specimens for immunocytochemical staining were prepared by a cell transfer method using archival Papanicolaou-stained imprint smears, and ER staining was performed by the labeled streptavidin method using an anti-ER monoclonal antibody. These results were compared with those obtained by DCC or EIA. In immunocytochemical staining for ER, positive staining was observed in the nuclei of tumor cells. A good correlation was obtained between the immunocytochemical staining results and biochemical results. Five cases were positive in anti-ER staining but negative in biochemical tests, and two cases were negative in anti-ER staining and positive in biochemical tests. Unlike biochemical assays, the immunocytochemical method does not necessitate use of fresh frozen materials and can be performed even using archival Papanicolaou-stained smears. Immunocytochemical study is a highly useful method for routine ER determination.

Reversal of estrogen‐resistance in murine mammary adenocarcinomas

From mouse mammary progestin-dependent (PD) adenocarcinomas induced with medroxyprogesterone acetate (MPA) we developed several in vivo lines that are maintained by subcutaneous syngeneic passages in MPA-treated mice and express estrogen (ER) and progesterone receptors (PR). Although most lines remained PD, with time some progestin-independent (PI) variants arose. Both PD and PI tumors regress with estrogen treatment although estrogen-resistant variants may also arise. The object of this paper was to investigate the reversibility of estrogen-resistance and its possible relation with hormone receptor down-regulation. Tumor regression was induced in a progestin-independent tumor line (BET) by treatment with a 5 mg 17beta-estradiol (E2) silastic pellet. One of the tumors started to grow disclosing an estrogen-resistant pattern of growth. This tumor line (BET-R) was transplanted into E2-treated and untreated animals (n = 4-6), selecting for the next passage tumors growing in treated animals. Seven new sublines were obtained at different passages by selecting for the next passage the tumors that had grown in untreated mice (BET-Ra-BET-Rg), until no tumors grew in E2-treated mice. ER and PR were measured by a ligand-binding, dextran-coated charcoal method using a single saturating point. From the seven sublines initiated, the first four proved to be reversible after 3-6 generation transplantation and the last three did not revert. A difference in PR expression between BET and BET-R (p < 0.05) was registered, but it did not correlate with the specific hormone behavior since two reverted lines had a pattern similar to that of BET and the other two were similar to BET-R. The expression of PR was higher in E2-treated mice (p < 0.05) and highly variable in the parental line. This led us to study the expression of PR at different stages of the estrous cycle. Higher levels of PR were observed in proestrous, estrous, and metestrous (p < 0.05) than in diestrous, and undetectable levels were found in ovariectomized mice. No differences in ER expression were detected during the estrous cycle. It can be concluded that under certain experimental conditions, estrogen-resistance is a reversible phenomenon. The experimental manipulation of hormone resistance may help develop strategies to modify the response to anti-hormones in humans.

Impact of Steroid Receptors, pS2 and Cathepsin D on the Outcome of N+ Postmenopausal Breast Cancer Patients Treated with Tamoxifen

In spite of the complexity of the biological basis of the hormonal regulation of breast cancer, clinical studies tend to simplify the information by mainly categorizing continuous variables related to hormonal status and not considering the interactions between variables. The present study was planned to examine the presence of an interaction between cathepsin D (Cath-D) and pS2 in patients treated with adjuvant tamoxifen in a homogeneous subset of node-positive postmenopausal patients and to evaluate the contribution of the interaction to the predictive ability of the model. Steroid receptors (ER and PgR) were measured in cytosol using the dextran-coated charcoal method, while Cath-D and pS2 were determined using commercially available immunoradiometric assays. The prognostic role of each variable and their joint effect were investigated using a Cox regression model. Biological variables were analyzed as continuous and when their prognostic relationship did not seem linear, a restricted cubic spline regression smoothing approach was adopted. The logarithm of hazard showed a linear relationship with the log(ER), while it i) remained almost constant up to about 20 fmol/mg and subsequently decreased for PgR; ii) was almost constant up to about 50 pmol/mg and subsequently decreased for Cath-D; iii) decreased for increasing log(value) up to about 33 ng/mg and subsequently increased for pS2. In the multivariate analysis both PgR and the interaction between pS2 and Cath-D retained a significant prognostic role. For low values of pS2, the prognosis worsened with the increase in Cath-D levels and this relationship reversed for high values of pS2. From the results of the present study we can conclude that i) a significant interaction between Cath-D and pS2 was found in this case series; ii) the prognostic relationship should not be underestimated in clinical decision making; iii) a predictive score obtained considering the contribution of PgR, pS2 and Cath-D could be useful for clinical use.