Dextran-coated Charcoal Assay Research Articles

An aptasensor for rapid and sensitive detection of estrogen receptor alpha in human breast cancer.

The analysis of estrogen receptor (ER) expression in breast carcinomas plays a crucial role in determining the endocrine responsiveness of tumors for systemic adjuvant therapy. Conventionally, the ER levels in breast carcinomas had been detected using the dextran-coated charcoal assay and radioimmunoassay, which are now substituted with safer and economic antibody-based assays such as immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). Despite a gold (Au) standard method, the IHC has been criticized for factors such as tissue fixation, antibody selection, and threshold staining for result interpretation that could falsify test accuracy and reproducibility. The quest for alternative methods of ER quantification in tissue samples paved the way for aptamer-based diagnostics. Previously, we have isolated a DNA aptamer against human ER alpha (ERα) using an in vitro evolution system. In this study, we developed an electrochemical sensor using the 76-nucleotide DNA ERα- aptamer for rapid, precise, and cost-effective detection of ERα expression in human breast cancer patients. The aptasensor was constructed by covalently immobilizing the thiolated ERα- aptamer onto a screen-printed Au electrode. Construction of aptasensors was confirmed through atomic force microscopy and differential pulse voltammetry measurements. A detection limit of 0.001 ng/ml was calculated for full-length ERα (66.2 kDa) in a detection time of 10 min. Analysis of the cancerous breast tissue samples using the ELISA and aptasensor methods enabled distinctive classification of samples into the categories of ER -ve, weak ER +ve, and strong ER +ve samples. The current change of this aptasensor lies within 5% after a storage of 60 days at 4°C. Further studies on a reasonably large sample size are required to realize the clinical potential of the sensor.

Molecular diagnostics for precision medicine in breast cancer treatment: what does the future hold?

Personalized medicine or precision medicine is one of the most interesting topics in current clinical cancer research. When compared to other types of solid cancer, breast cancer has been utilized sub-personalized (subtyping) treatment strategy for a very long time. In the 1980s, the presence of estrogen receptor (ER) was evaluated by a dextran-coated charcoal assay (ligand binding assay) and endocrine therapy was administered to specific patients with ER-positive breast cancer [1]. LHRH analog for premenopausal patients is another subtyping approach. However, recent technological advances in multi-gene assay and whole genome sequencing have made it possible to divide breast cancer subtyping into more detailed categories. In addition, classical RT-PCR technique for mRNA expression analysis was developed to digital PCR assay with high sensitivity. Based on these technological advances, we are trying to administer the right drug at the right time to the right patient. The development of molecular diagnostic tools is a key element for realizing precision and personalized medicine in clinical practice. These tools should be industrialized and approved by a regulatory agency. This special feature issue of Breast Cancer includes five review articles related to molecular diagnostics. Figure 1 shows the application of each molecular diagnostic in clinical breast cancer time course. For instance, multi-gene classifiers are used for prediction of risk of recurrence, and companion diagnostics are applied for choosing a specific drug for each patient. Sato et al. cover the basic knowledge of ‘‘genomic tumor evolution’’. Recent progress of comprehensive genome-wide analysis has revealed information regarding genomic mutations and rearrangements in each individual tumor [2]; however, during the process of tumor invasion, metastasis and treatment exposure, cancer genomes are dynamically changing and evolving. At this moment, no established technique exists for tracking this genomic tumor evolution in the living tumor of a patient, although liquid biopsy to detect circulating tumor cells and circulating cell-free DNA could be a promising approach. Naoi et al. reported multi-gene classifiers for predicting recurrence risk for early breast cancer patients [3]. In contrast to conventional biomarkers such as ER, PgR and HER2, multi-gene assay evaluates multiple target mRNA expressions at one time, providing a significant amount of information for treatment decision. Although many multigene assays have been developed to date, most success case is the Oncotype DX assay which calculates the recurrence score (RS) based on the expression of 21 genes using RNA extracted from FFPE tumor tissues. Very recently, the first survival data of the TAILORx study with Oncotype DX has been reported [4]. Early breast cancer patients with ERpositive HER2-negative node-negative tumors and those whose tumors were classified as low RS (equal or less than 10) showed remarkable good prognosis without adjuvant chemotherapy. This is the first prospective evidence for treatment decision to have been made using multi-gene assay. Tazawa reported on the development of companion diagnostics for indication of molecular targeting drugs [5]. Though many molecular targeting drugs for breast cancer have been developed to date, the most scene-changing drug has been trastuzumab for HER2-positive tumor. Trastuzumab was developed through clinical trials that & Shigehira Saji ssaji@fmu.ac.jp

Histopathologic Prognosis of Thymomas

Particularly in anatomic pathology, the past 2 decades have witnessed a proliferation of tests that are surrogates for selected analytes of predictive or prognostic interest. Those targets include such moieties as functional hormone receptor proteins and the composition of genes that govern responses to selected growth factors. Common respective examples of those groups are estrogen and progesterone receptor proteins1 and the HER-2/ neu , c- kit , and epidermal growth factor receptor ( EGFR ) genes.2–8 Thousands of tests are done each year, predominantly through immunohistochemical analysis, to assess the status of such targets in malignant human neoplasms. And yet, immunohistochemical analysis represents an imperfect substitutive method in reference to the goal just mentioned. Immunohistology does not necessarily detect functional hormonal receptors (HRs),9 and it is suboptimally effective in separating HR-“positive” from HR-“negative” tumors, when compared with the performance of traditional biochemical HR assays.10 General assumptions also have been made (and accepted) that hold that the increased cellular expression of the proteins related to the neu , c- kit , and EGFR genes—as visualized by immunohistochemical analysis—is an invariable reflection of gene amplification or an activating mutation.11 Recent publications have demonstrated the fallacy of those suppositions. Increasingly, the published literature shows that fundamental, “firsthand” laboratory tests are incontrovertibly superior to immunohistochemical analysis as medical devices.8,12 Pertinent examples are the dextran-coated charcoal assay (DCCA) for HRs13 and in situ hybridization or polymerase chain reaction–based blotting techniques …

Preliminary results of postoperative adjuvant treatment with tamoxifen in colorectal cancer

To investigate estrogen receptor (ER) expression and the effects of anti-estrogen therapy on the prognosis of colorectal carcinoma. ER was measured in fresh colorectal cancer tissues by Dextran-coated charcoal (DCC) assay. The relationships between ER expression and clinicopathological parameters in colorectal cancer were analyzed. Tamoxifen was administrated postoperatively as adjuvant treatment. The positive rate of ER in colorectal tumor tissues was 37.0%. The 5-year survival rates of tamoxifen group and control group were 66.7% and 72.5% respectively, and there was no significant difference between the two groups. The distant metastasis rate of Tamoxifen group was significantly lower than that of control group (3% versus 20%). Some colorectal carcinomas are hormone-dependent tumors, and anti-estrogen therapy has no effect on them.

Medical thoracoscopy: hormone receptor content in pleural metastases due to breast cancer

Pleural metastases are common in the course of breast cancer, but, to date, the role of oestrogen receptor (OR) and progesterone receptor (PgR) content in metastatic tissue has been poorly evaluated. A series of 50 consecutive patients with a history of breast cancer (median age 64 yrs, range 40-86 yrs), which presented with pleural effusion and therefore underwent medical thoracoscopy, was analysed. Metastatic pleural involvement was histologically confirmed in all patients. The hormone receptor status of the pleural metastases was investigated using the immunohistochemical method in 49 and the biochemical method in 31 cases. The immunohistochemical test was performed using monoclonal antibodies. Biochemical analysis was performed on specimens quick-frozen in liquid nitrogen. OR and PgR were measured with the dextran-coated charcoal assay and Scatchard analysis. Immunohistochemical analysis yielded 29 OR-positive and 25 PgR-positive cases and biochemical analysis yielded 16 OR-positive and four PgR-positive cases, sometimes discrepant to hormone status of the primary breast cancer. Using a semiquantitative immunoreactive score, there was a significant association between receptor positivity and survival, but only for PgR positivity. Immunohistochemical and biochemical detection of hormone receptors (oestrogen and progesterone) in pleural metastases of breast cancer is feasible based on medical thoracoscopy as the method of choice, by which sufficient specimens may be obtained. The receptor status may enable a decision on antihormonal treatment. Whether a positive receptor status in pleural metastatic tissue is associated with a better prognosis remains to be confirmed.

Cytologic Diagnosis of Estrogen and Progesterone Receptors in Breast Imprints

To investigate estrogen receptor (ER) and progesterone receptor (PR) levels in imprint specimens obtained at breast surgery and to compare their correlation with that of standard methods. Imprint specimens for cytology were obtained from 101 mass-forming lesions in 66 patients, and specimens were frozen in liquid nitrogen for later assay. The imprint specimens were immunocytochemically (ICC) stained by monoclonal antibody to ER or PR; diaminobenzidine-stained cell nuclei in clusters were regarded as positive. Tissue specimens were assayed by the standard method of dextran-coated charcoal assay (DCC) and enzyme immunoassay. Forty-five primary breast cancer lesions, 2 contralateral breast cancer, 49 dissected nodes and 5 benign breast lesions were collected. The correlation between DCC and ICC was 81% (82/101) for ER and 74% (66/101) for PR. That between EIA and ICC was 88% (88/99) for ER and 80% (79/100) for PR, higher than that between DCC and ICC for ER and PR. ICC assessment of ER or PR on imprint cytology is a promising clinical test with an acceptable correlation.

Factors influencing the prognostic role of oestrogen and progesterone receptor levels in breast cancer—results of the analysis of 670 patients with 11 years of follow-up

In the last two decades, the prognostic role of the steroid hormone receptors has been the subject of a myriad of publications. Nevertheless, its relevance after long-term follow-up is still not clear. The confusion about the prognostic value is mainly due to the difficulty in comparing analyses. Despite different study-designs and statistical approaches, oestrogen (ER) and progesterone (PR) receptors are widely accepted as prognostic factors. Data from 670 breast cancer patients with a median follow-up of 11.4 years were analysed retrospectively. ER and PR were measured by the dextran-coated charcoal (DCC) assay. To investigate the time dependence of the prognostic relevance of ER and PR, separate analyses were done for follow-up shorter and longer than 5 years. Special focus was directed at patients ⩽50 and >50 years, node-negative women, in particular those without adjuvant therapy. Univariate and multivariate analyses were performed. In univariate analysis, ER and PR were associated with a significantly longer overall survival at the cut-off levels 10, 20 or 100 fmol/mg protein. The significant survival benefit occurred in the first 5 years of follow-up and remained unchanged in the following period. In the multivariate analyses, only the PR was of significant prognostic value (for PR⩾20 fmol/mg P=0.036, for PR⩾100 P=0.01, Cox analysis). In patients younger than 51 years, only PR was an independent prognosticator at the cut-off level of 100 fmol/mg protein, while in patients >50 years both hormone receptors were not significant. In N0 patients, only the PR reached long-term prognostic independence at a cut-off point of ⩾100 fmol/mg (P=0.018). In addition, in the group of node-negative women ⩽50 years without adjuvant therapy the PR level reached prognostic significance. The hormone receptor status was a prognostic factor only during the first 5 years of follow-up. Our data suggest that age, lymph node status, length of follow-up and probably the ER/PR assay are important for the evaluation of ER and PR as prognostic variables. In most analyses, PR appeared to be superior to ER in predicting the prognosis of primary breast cancer patients.

The immunocytochemical versus cytosol measurement of the oestrogen receptor in invasive breast cancer tissue

To compare two methods of measurement of oestrogen receptor (ER)-expression in invasive breast cancer tissue. Sections from 299 breast cancer cases were stained for the ER by immunocytochemical assay (ICA), using mouse monoclonal antibody (MAb) NCL-ER-6SF11, and by the dextran-coated charcoal assay (DCC). Concordant results were observed in 230 of the 299 cases (77%), 69 patients had discordant results (kappa=0.537). We found a moderate concordance between ICA and DCC for ER measurement in breast cancer tissue. If we change the golden standard from DCC to ICA, 23% of patients would receive a different therapeutic approach.

Immunohistochemical assay for detecting estrogen receptors in canine mammary tumors

Abstract Objective To determine the estrogen receptor (ER) content of canine mammary gland tumors by use of immunohistochemical (IHC) examination of formalin-fixed sections. Sample Population 21 mammary gland tumors from 20 adult dogs. Procedure ER were detected in formalin-fixed tissues, using an avidin-biotin alkaline phosphatase IHC assay and were quantified on fresh-frozen tumor samples, using a modified dextran-coated charcoal (DCC) assay. Results 7 of 21 tumors had visually detectable nuclear ER by use of IHC staining, whereas 8 of 21 tumors were positive for ER by use of the DCC assay. The ER-positive cells in 5 IHC-positive tumors were epithelial cells with histologic criteria of early malignancy. The remaining 2 ER-positive tumors detected by use of IHC had ER-positive mast cells within areas of connective tissue around the tumor. Conclusions Immunohistochemistry is an additional method for detection of ER in canine mammary tumors. The major advantage of this type of assay is that it may be performed on formalin-fixed tissues, and individual ER-positive cells may be identified. Discovery of ER-positive mast cells by use of IHC is of concern, particularly if the ER status of a tumor is based on DCC results alone. Clinical Relevance Because most canine mammary tumors are fixed in formalin prior to histologic evaluation, an IHC assay that identifies ER-positive cells is desirable. Adjunctive antiestrogen therapy could be administered to dogs with ER-positive tumors. (Am J Vet Res 1999;60:627–630)

Relationship between steroid receptors (as continuous variables) and response to adjuvant treatments in postmenopausal women with node-positive breast cancer.

In current clinical practice for breast cancer patients, estrogen (ER) and progesterone receptor (PgR) concentrations, quantified by the dextran-coated charcoal assay, are categorized by an arbitrary cutoff into a negative or positive status. However, although the results obtained with this approach are easy to interpret, such a representation could oversimplify the relationship between ER and PgR content and patient outcome and imply an assumption of monotonicity, which is generally expected but rarely proven. We evaluated the relationship between ER and PgR content (considered on a continuous scale) and clinical outcome, using a flexible statistical model, in a group of postmenopausal patients with N-positive operable tumors who were submitted to surgery and different adjuvant treatments (tamoxifen or CMF). Univariate analysis indicated that in the tamoxifen-treated group, ER level, number of metastatic nodes (pN) and age, but not PgR, were significant indicators of clinical outcome (p = 0.032, p = 0.021 and p = 0.029, respectively). Multivariate analysis indicated that in this group of patients there was no interaction between variables, and in the final model for disease-free survival (DFS) only ER and pN were retained with an overall predictive ability of the regression model of 0.723, as evaluated by Harrell's c. However, pN markedly contributed to the predictive ability of the model with respect to ER, since a marked decrease in Harrell's c statistic (c = 0.582) was observed when pN was removed from the model. In the CMF-treated group, only pN affected clinical outcome. When the estimated DFS curves obtained from the final Cox regression models were plotted according to four values of ER (in the tamoxifen-treated group) or three values of pN (in the CMF-treated group) we observed that in the tamoxifen-treated group patients with an ER concentration equal to 0 fmol/mg cytosol protein had the worst prognosis, whereas a marked improvement of the expected DFS was observed for patients with a low but detectable ER level (generally classified as ER-negative because falling below the conventional cutoff value of 10 fmol/mg cytosol protein). Our results seem to suggest that the use of steroid receptor concentrations on a continuous scale, instead of dichotomous "status", is to be preferred in the choice of adequate therapeutic strategies.

Paraffin section immunocytochemistry and cytosol-based ligand-binding assays for ER and PR detection in breast cancer: the time has come for more objectivity

The importance of the receptor level in breast cancer as an indicator of hormone response has been extensively studied for more than 20 years. Besides cytosol-based ligand-binding assays (dextran-coated charcoal assay, DCC), new methods using monoclonal antibodies raised against estrogen and progesterone receptors allow for the detection of receptors both in cytosol extracts (enzyme immunoassay, EIA) and in tissue sections (immunocytochemical assay, ICA). The biochemical assays (DCC and EIA) as well as the immunochemical detection (ICA) have specific qualities and produce original information which is useful for the therapeutic decision. While DCC gives a measure of the receptor level, whatever the real source of synthesis (normal and/or neoplastic tissue), ICA locates the positive cells and their relative proportion in the tumor. Both methods present their own advantages and disadvantages which are summarized in this study.

Influence of Fixation, Antibody Clones, and Signal Amplification on Steroid Receptor Analysis

: Various factors influence analysis of steroid receptors, including tissue processing, fixation, and antibody clones used. Furthermore, the detection methods, that is, use of epitope retrieval by microwave heating and signal amplification, may affect the reliability of the results. Eleven different fixation/tissue-processing methods as well as different commercial antibodies for the estrogen-and progesterone-receptors and the tyramine amplification technique were evaluated for optimizing the staining procedures. Moreover, the results from the optimal immunohistochemical procedure were compared with results obtained using a biochemical dextran-coated charcoal assay in 88 breast carcinomas from postmenopausal patients. The four antibody clones tested (1D5, 6F11, 1A6, and antiserum) revealed different sensibility to formalin, either favoring shorter or longer periods of fixation. Overall, a delay in the onset of fixation showed the worst results, whereas overfixation for up to 4 days or pH of formalin had little influence. Using the new tyramine technique it was possible to reduce costs per slide considerably by increasing antibody dilution. Comparison of the biochemical assay with optimized immunohistochemistry in the 88 cases investigated showed an overall concordance of 98.9%. Our study suggests an optimized immunohistochemical procedure that can replace the biochemical “gold standard” if standardization of tissue processing is maintained.

Long-term Prognostic Impact of Immunohistochemical Estrogen Receptor Determinations Compared with Biochemical Receptor Determination in Primary Breast Cancer

The estrogen receptor content of primary breast cancer is predictive for the outcome and response to hormonal manipulation ( I ) . Most data on the prognostic implication of estrogen receptor determinations have been generated with biochemical receptor determinations utilising the dextran-coated charcoal (DCC) method and more recently the RIA assay (2). During the last decade immunohistochemical (IH) receptor determinations have gained in popularity due to their lower costs and the lower requirements for sample size as well as the possibility to perform the determination on archival tumour material. A large number of publications have demonstrated a good correlation between IH and biochemical receptor determinations, but also clearly discrepant findings have been reported (for a review see (2)). Few studies have assessed the clinical utility of IH. A number of studies have shown that the predictive value of IH receptor determinations for endocrine treatment response are at least as good as those of biochemical assays for estrogen receptor binding (2). Only a few studies have compared the prognostic value of IH and biochemical estrogen receptor determinations (3-5). The purpose of the present investigation was to study the correlation between IH and DCC estrogen receptor determination as well as their prognostic value for recurrence and death. Material und Methods. A total of 142 consecutive cases of invasive breast cancer analysed at the Department of Pathology, University of Helsinki, during the years 1987-1988, were included in the study. The distribution of TNM-classes and the proportion of estrogen receptor positivity are shown in the Table. In 9 cases the determination was performed on a local recurrence. These patients are excluded from the analysis on the prognostic value of receptor determinations. One patient with previous metastatic contralateral breast cancer was excluded from analysis of survival and disease-free survival. Other patients with synchronous or metachronous bilateral breast cancer (n = 18) were included in all the analyses. In 5 cases TN-status was not known, all were cases with receptor determinations from a local recurrence. The turnour samples were sent to the laboratory on ice and arrived unfixed to the histological laboratory for diagnosis and estrogen receptor (ER) staining. Immunohistochemical staining for ER was performed on frozen sections using the ER-ICA kit (Abbott Laboratories, Chicago, IL, USA). The sections were fixed and stained for ER following the manufacturer’s directions, counterstained lightly in Mayer’s hematoxylin and mounted. The percentage of positively stained turnour cell nuclei was estimated by counting 5001 000 morphologically malignant cells in multiple random fields in every sample. The turnours were divided into four categories according to the number of positive nuclei found: 0-9% = negative, 10-39% = weakly positive (+), 40-69% moderately positive (+ +) and 70% or more = strongly positive ( + + +). Only nuclear immunohistochemical staining was regarded as positive. A sample of each tumour was taken by the surgeon, immediately frozen in liquid nitrogen and analysed at the Department of Clinical Chemistry for cytosol ER by the dextran-coated charcoal method (6) . Cause-specific disease-free survival (DFS) and cause-specific overall survival (0s) were estimated by the Kaplan-Meier method, and the statistical significance of differences tested by the log-rank test. For analysis of both 0s and DFS only breast cancer-related deaths were classified as events, i.e., patients dying from other causes without breast cancer recurrences were classified as censored at the time of death. Follow-up times ranged from 7 to 9 years with a median of 8 years. Fifty-eight patients (42%) had TI tumours, 62 (45%) T2, 12 (9%) T3 and 4 (4%) T4 tumours. Sixty-two patients (45%)) had axillary metastases. Only one patient had distant metastases at diagnosis. Fifty-four patients have died, 13 from causes other than breast cancer without evidence of recurrence. Results. The correlation between IH ER score and ER positivity by DCC assay was studied using a cut-off point of 10 fmol/rng protein (Table). In 44 cases (31%) the result was discrepant. Thirty-three cases (23%) were IH positive and DCC negative, whereas 11 cases (8%) were IH negative and DCC positive. The

Comparison of estrogen receptor determinations by a biochemical ligand‐binding assay and immunohistochemical staining with monoclonal antibody ER1D5 in females with lymph node positive breast carcinoma entered on two prospective clinical trials

BACKGROUND The measurement of estrogen receptors (ER) in breast cancer specimens has traditionally been assessed with a dextran-coated charcoal assay (DCCA). More recently the immunohistochemical staining (IHC) method has gained increasing popularity because of its ability to use fixed tissue, assess needle biopsies, and reduce cost. Controversy exists over the accuracy of IHC compared with that of DCCA in determining ER. We compared these two techniques using tumor tissue obtained from a large group of females with lymph node positive breast carcinoma with long term follow-up. METHODS Breast carcinoma tissue was obtained from a large group of females with node positive breast carcinoma participating in two adjuvant chemotherapy trials. ER was determined by the traditional DCCA method and by IHC using the ER1D5 antibody. Disease free survival (DFS) and overall survival (OS) were assessed by each of these methods. RESULTS ER status was determined by DCCA and IHC in tumor tissue obtained from 316 females. A concordance of 79% was observed for the determination of ER-positive tumors. Of the discordant results, the majority of DCCA-negative, IHC-positive tumors could be explained by a low level of DCCA positivity (<10 fmol) or IHC staining of nonmalignant cells. A much higher rate of discordant results was observed in premenopausal females. Of the DCCA-negative, IHC-positive patients 97% were premenopausal and of the DCCA-positive, IHC-negative patients 79% were premenopausal. ER by DCC appears to perform better than ER by IHC as a prognostic factor in terms of DFS and OS. CONCLUSIONS When compared with DCCA, IHC with monoclonal antibody ER1D5 appears to be a reasonable substitute for the determination of ER. Although DCCA appeared to perform better as a determinant of prognosis, ER detection is used primarily for deciding on hormonal therapy. Review of discordant cases indicates IHC may more accurately reflect the ER status of malignant cells in some patients. Attention must be paid to quality control considerations in performance of IHC staining. Cancer 1996;78:764-72.

Purification and reassessment of ligand binding by the recombinant, putative juvenile hormone receptor of the tobacco hornworm,Manduca sexta

The 29 kDa protein from the larval epidermis of the tobacco hornworm, Manduca sexta, that specifically bound photoaffinity analogs of JH I and JH II was produced by a recombinant baculovirus (rJP29). The higher of the two molecular weight forms made corresponded to a protein that could be formed by read-through of the TGA termination codon to the following TAA. The previously reported, apparent high affinity binding of [methyl-3H]-JH I by rJP29 as measured by the dextran-coated charcoal (DCC) assay [Palli et al., Proc Natl Acad Sci USA 91:6191–6195 (1994)] was found to be artifactual due to endogenous cellular esterases that co-purified with rJP29 through both DEAE cellulose and MonoQ chromatography. These esterases converted the 10–20 nM labelled JH to JH I acid and [3H]-methanol during the 1 h incubation at room temperature. Additionally, DEAE fractions containing rJP29 or from wild-type virus-infected cells were found to bind nonspecifically high amounts of 12, 13-3H]-JH I acid in the DCC assay. Neither rJP29 nor the cellular esterases had JH esterase activity when assayed on a series of thioether surrogate substrates. When separated from these contaminating esterases either by hydroxylapatite or affinity chromatography, rJP29 showed little or no detectable binding of [12,13-3H]-JH I. Yet purified rJP29 bound EBDA, the photoaffinity analog of JH I; and this binding was prevented by retinol, JH I, methyl farnesoate, methoprene, and xanthophyll, but not by farnesol and 20-hydroxyecdysone. Therefore, JP29 is not a high affinity JH receptor. © 1996 Wiley-Liss, Inc.

Immunohistochemical Detection of Estrogen and Progesterone Receptors Performed with an Antigen-Retrieval Technique on Methacarn-Fixed Paraffin-Embedded Breast Cancer Tissues

Immunohistochemical detection of estrogen and progesterone receptors (ER and PgR, respectively) was performed in 67 cases of Japanese female invasive breast carcinoma on methacarn-fixed paraffin-embedded sections using monoclonal antibodies against ER (1D5) and PgR (10A9) with an antigen-retrieval technique based on microwave exposure in citrate buffer solution. Staining localized in nuclei and specimens containing >=20% specificially stained tumor cell nuclei were considered ER- or PgR-positive; the positive rate was 37% (25/67) for ER and 45% (30/67) for PgR. The immunohistochemical (IHC) results were compared with cytosolic receptors obtained from tissue homogenates as measured by dextran-coated charcoal (DCC) assay, in which tumors were considered ER- and/or PgR-positive when receptor concentration was greater than 3 or 5 fmol/mg cytosolic protein, respectively, and agreement between the two methods was 71.6% for ER and 80.6% for PgR. The sensitivity and specificity were 53.5 and 91.7% for ER-IHC, and 77.4 and 83.3% for PgR-IHC, respectively. In relation to age of the patient and stage of the cancer, ER immunoreactivity correlated with patient's age (>=55 vs 55,P= 0.032), and proportionally increased with aging (P= 0.0084) using the Kruskal–Wallis statistics. PgR-immunoreactivity correlated with nodal involvement (P= 0.031) by the TNM system. However, no correlations were found between the results of the DCC assay and any of the clinical parameters examined. Thus, immunohistochemical assay may provide valuable information in predicting prognosis and response to endocrine therapy.

Estrogen Receptor Determinations of Cutaneous Recurrences of Breast Cancers: Reduced Sensitivity of Hormone Binding Assays

: Patients with estrogen receptor positive breast carcinoma occasionally develop cutaneous recurrences that apparently do not contain receptors when analyzed by the dextran coated charcoal method. Since these nodules may contain only a few tumor cells, we wondered if such negative results were accurate. Our study group consisted of 19 pairs of specimens, each pair comprising a cutaneous nodule and the primary tumor from which it arose. Estrogen receptor determinations using the dextran coated charcoal method had been performed on each cutaneous specimen, and we repeated these determinations using an immunohistochemical method. We then compared the data derived from the cutaneous specimens with the estrogen receptor status of the corresponding primary tumors. The dextran coated charcoal method failed to detect receptors in about 70% of the recurrences that arose from receptor positive tumors (10 of 14 cases). The immunohistochemical staining, on the other hand, detected estrogen receptor protein in all 14 of these cutaneous nodules. In six cases, the small number of tumor cells in the cutaneous specimens could explain the discrepancy between the results of the immunohistochemical staining and the hormone binding assay. Administration of tamoxifen probably accounts for the divergent results in one other case. In the final three cases, the discrepancies remain unexplained. We conclude that cutaneous recurrences of breast carcinoma display estrogen receptors about as frequently as do primary tumors, but that the dextran coated charcoal assay may not detect the receptors in some cutaneous specimens. Physicians should bear these observations in mind when they encounter a case in which an estrogen receptor positive tumor has apparently given rise to a receptor negative cutaneous recurrence. Immunohistochemical staining of the nodule might reveal the receptors in such a case.

Steroid hormone receptors in three human gastric cancer cell lines.

Steroid hormone receptors in three human gastric adenocarcinoma cell lines and their transplanted tumors (except nontumorigenic KATO-III) in nude mice were determined by dextran-coated charcoal assay. Progesterone receptors (PgR) were found in all cell lines, transplanted NUGC-3, and AZ 521 tumors. Estrogen receptors (ER) were found in KATO-III cells, transplanted NUGC-3, and AZ 521 tumors, whereas glucocorticoid receptors (GR) were found only in NUGC-3 tumor and no androgen receptor was found in any cell lines or transplanted tumors. Since NUGC-3 cells had ER, PgR, and GR, it was used for the study of the effects of steroid hormones on growth. The results showed the cell cycle phase distributions and growth rate of transplanted tumors were similar in hormone-treated and nontreated groups. The persistent expression of PgR in gastric cancer cell lines and tumors, and the slight increase of tumor volumes in the progesterone-treated group suggests that progesterone and its receptors may be important in the pathogenesis of gastric cancer, but their biological function remains to be elucidated.

Oestrogen receptors in endometrial cytosol of gilts on days 10-13 of oestrous cycle and pregnancy

Cytosolic oestrogen receptors (ER(c)) were determined with a Dextran-coated charcoal assay and Scatchard plot analysis in endometrial tissue of non-pregnant and pregnant gilts on Days 10-13 after standing oestrus. The Kd of the ER(c) was 0.40 +/- 0.04 nM (mean +/- SEM) and was not affected by day or reproductive state. The endometrial ER(c) concentration was affected by day and reproductive state (P

Determination of estrogen receptors in paraffin‐embedded tissue: Techniques and the value in breast cancer treatment

Estrogen receptor (ER) analysis in breast cancer has been used in three clinical situations: to select patients with advanced breast cancer for hormonal therapy, as a prognostic parameter, and for selection of women with early breast cancer to adjuvant hormonal treatment. ER has traditionally been measured using labelled hormone in binding assays—often in dextran-coated charcoal assays (DCC). Monoclonal antibodies to ER has permitted development of a solid phase enzyme immunoassay (ER-EIA) used for quantitative determination of ER in tissue homogenates, and have also been used for determination of ER using an immunohistochemical assay in frozen sections (ER-ICA) or in formalin-fixed, paraffin-embedded tissue (ER-PAR). A large number of studies has compared ER-EIA with ER-DCC assays. There is a good linear correlation between the two types of assay but ER-EIA measure more ER and classify a larger fraction of tumors ER-positive than conventional ER assays. Lack of clinical data makes the significance of thi...