Sources/Clones
Abbott (H222), Accurate (CC4.5), Biogenesis (ERLH1), Dako (1D5), Eurodiagnostics/Accurate (polyclonal), Immunotech (1D5), Novocastra (CC4.5, LH1), Oncogene (TE111), Vector (6F11) and Zymed (iD5).
Fixation/Preparation
The method of preparation is very much dependent on the antibody clone employed. The ERICA antibody (clone H222) is mostly only immunoreactive in fresh-frozen tissues although some laboratories had success with tissues which had only short exposure to formaldehyde (Raymond & Leong, 1988, 1990) or following the careful use of specific antigen retrieval agents such as DNAse. The development of clone 1D5 made it possible for the immunostaining of routinely fixed paraffin-embedded sections but only following heat-induced epitope retrieval (HIER) (Leong & Milios, 1993; Balaton et al, 1996). The latter procedure has no effect with the H222 antibody.
Background
The first monoclonal antibodies to the estrogen receptor (ER) protein (estrophilin) were produced from a human breast cancer cell line, MCF-7, subjected to affinity column processing and elution (Greene et al, 1980). The antibodies were produced by immunization of rats with this partially purified estradiol-estrophilin complex. Fusion of splenic lymphocytes from the immunized animals with myeloma cells yielded three hybridoma cells lines after cloning by limited dilution techniques, the antibodies thus produced recognized estrogen-occupied as well as unoccupied receptors (Pousen et al, 1985).
The human ER is a member of a family of nuclear receptors for small hydrophobic ligands such as thyroid hormone, vitamin D, retinoic acid and the steroid hormones. Each receptor has a ligand-binding domain, a hinge region, a DNA-binding domain and a variable or regulatory domain. The ER gene is located on the long arm of chromosome 6 (q24-27) and comprises eight exons and intervening introns spanning at least 140 kilobases. Binding of the ligand to the receptor is thought to result in an allosteric alteration that allows the hormone-receptor complex to bind to its DNA response element in the promoter region of a target gene. In the absence of hormone binding, the domain appears to be inhibitory in function, preventing transcriptional activation. Besides establishing the allosteric association of the hormone binding and the regulatory domains, the sequences contained in the hinge region are critical in directing the ER and progesterone (PR) proteins to the nucleus after they are synthesized in the cytoplasm. The DNA-binding domain has many basic amino acids, some of which are repeating units folded into a ''fingered" structure coordinated by a zinc ion, known as the "zinc finger" (Schwabe et al, 1990). The ER and PR appear to enhance the transcriptional activity of selected genes. The actual mechanism is not known but probably involves interactions between receptors and other transcriptional factors with the promoter regions of the respective genes. In the current model of ER action, estradiol diffuses into the cell and binds to the receptor, leading to its dimerization and tight binding to its specific DNA target. Following this binding to the estrogen response element in target genes, there is stimulation to increase the transcription of target genes, some directly or indirectly leading to the establishment of both autocrine and paracrine growth stimulatory loops (Leong & Lee, 1995).
Early studies employing monoclonal antibodies to estrophilin reported both cytoplasmic and nuclear staining, the former being stronger. However, subsequent studies have established an exclusive nuclear localization for ER proteins in both human breast carcinomas as well as other steroid-responsive tissues. This has been confirmed by autoradiographic studies and immunoelectron microscopy has shown the ER protein to be present in the euchromatin portion of the nucleus in breast, endometrial and ovarian cancers as well as in benign endometrium. The cytoplasm in all cases did not reveal presence of the receptor although there was some reaction product in the ribosomes. This latter reactivity was considered to be non-specific although the possibility of synthesis of ER at the ribosomal level was not completely ruled out. Studies demonstrating cytoplasmic localization of ER protein in addition to nuclear localization have mostly employed fluoresceintagged estrogen analogs, whereas modern immunoenzyme techniques utilizing monoclonal antibodies to ER protein have shown only nuclear reactivity.
Applications
Over 100 years ago it was recognized that oophorectomy was associated with clinical remission in women with metastatic breast cancer. Despite the usefulness of hormonal manipulation in some women, only approximately 30% of unselected women with metastatic breast cancer responded to such treatment. There was therefore a need to distinguish those women whose breast cancers are hormone dependent from those whose tumors are hormone independent. Employing cytosol-based ligand-binding assays, it was shown that about 50-60% of women with ER-rich breast cancers responded to hormone treatment, while less than 10% with ER-poor tumors showed a similar response. The relevance of ER status and hormonal treatment in node negative tumors, however, is less clear. It is also clinically recognized that a small proportion of patients whose tumors are receptor negative by cytosol-based assays will show a positive response to hormone treatment and as many as one-third of those with ER-positive tumors may fail to respond to such treatment. On the basis of comparative immunohistologic studies, it is believed that some of these discrepancies are caused by inherent errors of the biochemical method which assays homogenized tissue samples with resultant errors introduced by the inclusion of benign epithelium, the dilutional effect of abundant stroma and inadequate tumor sampling. Indeed, there is recent persuasive evidence, based on hormonal response as the ultimate yardstick, that immunostaining in frozen or paraffin sections is the more accurate measurement of ER status (Pertschuk et al, 1996) and this and many other advantages make immunostaining the "gold standard" to replace cytosol assays (Taylor, 1997).
It has been suggested that ER may be used to identify metastatic breast carcinoma but a variety of other lesions with epithelioid features may also express ER.
These include epithelioid smooth muscle tumors, malignant melanoma, meningioma, sclerosing hemangioma (Leong et al, 1997), desmoid tumors, thyroid neoplasms and cervical, endometrial and ovarian cancers, rendering the marker less useful as a diagnostic discriminant.
Comments
Since the development of clone 1D5, it has become possible to perform immunostaining for ER in routinely fixed and processed tissues inexpensively, accurately and consistently. Some form of HIER procedure is essential when using clone 1D5; in contrast, H222 fails to stain following HIER, indicating that only one epitope of the ER, and not the entire antigen, is retrieved by the heating process. For this reason, it has been suggested that the procedure be called heat-induced epitope retrieval rather than the original term "heat-induced antigen retrieval". It is possible to obtain consistent staining of ER in cytologic preparations by employing clone 1D5 with HIER on smears which are initially completely air-dried before fixation in 10% buffered formalin.
It is important, as with most other antibodies employed for immunohistology, that each laboratory determines its optimal time for epitope retrieval and does not purely rely on the procedures developed for other laboratories. determines its optimal time for epitope retrieval and does not purely rely on the procedures developed for other laboratories.
A concordance of 77-100% exists between immunostaining in paraffin and frozen sections with the dextran-coated charcoal assay (DCC) and we have adopted 10% staining of tumors cells as the cut-off value as it corresponded with 10 fmol/mg of proteins by DCC assay. There is strong correlation-of the percentage of positive cells with the intensity of staining. The results of immunostaining may be expressed subjectively as positive or negative, as a percentage of positive tumor cells, or as a score derived by adding grades of staining (1, 2 and 3, corresponding to mild, intermediate and strong staining) and the percentage as scores of 0, 1, 2, 3 and 4 (corresponding to 10%, 11-25%, 26-50%, 51-75% and > 75%). Computerized image analysis has been claimed to produce an increasing specificity and sensitivity relative to biochemical assays but other studies have shown identical results by image analysis and visual examination and significantly similar agreement between the two and biochemical values.
References
•Balaton AJ, Mathieu M-C, Le Doussal V 1996 Optimization of heat-induced epitope retrieval for estrogen receptor determination by immunohistochemistry on paraffin sections. Results of a multicentric comparative study. Applied Immunohistochemistry 4: 259-263.
•El-Badawy N, Cohen C, De Rose PB, Sgoutas D 1991 Immunohistochemical estrogen receptor assay: quantitation by image analysis. Modern Pathology 4: 30-309.
•Greene GL, Fitch FW, Jensen EV 1980 Monoclonal antibodies to estrophilin: probe for the study of estrogen receptors. Proceedings of the National Academy of Sciences USA 77: 157-161.
•Leong AS-Y, Lee AKC 1995 Biological indices in the assessment of breast cancer. Molecular Pathology 48: M221-M238.
•Leong AS-Y, Milios J 1993 Comparison of antibodies to estrogen and progesterone receptors and the influence of microwave-antigen retrieval. Applied Immunohistochemistry 1: 282-288.
•Leong AS-Y, Chan KW, Leong FJ 1997 Sclerosing hemangioma. In: Corrin B (ed) Pathology of lung tumors. London: Churchill Livingstone, pp 175-188.
•Pertschuk L, Feldman J, Kim Y-D et al 1996 Estrogen receptor (ER) immunocytochemistry in paraffin with ER 1D5 predicts breast cancer endocrine response more accurately than H222Sp in frozen sections or cytosol-based ligand binding assays. Cancer 77: 2541-2549.
•Pousen HS, Ozello L, King WJ, Greene GL 1995 The use of monoclonal antibodies to estrogen receptors (ER) for immunoperoxidase detection of ER in paraffin sections of human breast cancer tissue. Journal of Histochemistry and Cytochemistry 33: 87-92.
•Raymond WA, Leong AS-Y 1988 An evaluation of potentially suitable fixatives for immunoperoxidase staining of estrogen receptors in imprints and frozen sections of breast carcinoma. Pathology 20: 320-325.
•Raymond WA, Leong AS-Y 1990 Estrogen receptor staining of paraffin-embedded breast carcinomas following short fixation in formalin: a comparison with cytosolic and frozen section receptor analyses. Journal of Pathology 160: 295-303.
•Schwabe JWR, Newhause D, Rhodes D 1990 Solution structure of the DNA-binding domain of estrogen receptor. Nature 348: 458-461.
•Taylor CR 1997 Paraffin section immunocytochemistry for estrogen receptor. The time has come. Journal of Histotechnology 20: 97-100.
Bibliografía
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
Abbott (H222), Accurate (CC4.5), Biogenesis (ERLH1), Dako (1D5), Eurodiagnostics/Accurate (polyclonal), Immunotech (1D5), Novocastra (CC4.5, LH1), Oncogene (TE111), Vector (6F11) and Zymed (iD5).
Fixation/Preparation
The method of preparation is very much dependent on the antibody clone employed. The ERICA antibody (clone H222) is mostly only immunoreactive in fresh-frozen tissues although some laboratories had success with tissues which had only short exposure to formaldehyde (Raymond & Leong, 1988, 1990) or following the careful use of specific antigen retrieval agents such as DNAse. The development of clone 1D5 made it possible for the immunostaining of routinely fixed paraffin-embedded sections but only following heat-induced epitope retrieval (HIER) (Leong & Milios, 1993; Balaton et al, 1996). The latter procedure has no effect with the H222 antibody.
Background
The first monoclonal antibodies to the estrogen receptor (ER) protein (estrophilin) were produced from a human breast cancer cell line, MCF-7, subjected to affinity column processing and elution (Greene et al, 1980). The antibodies were produced by immunization of rats with this partially purified estradiol-estrophilin complex. Fusion of splenic lymphocytes from the immunized animals with myeloma cells yielded three hybridoma cells lines after cloning by limited dilution techniques, the antibodies thus produced recognized estrogen-occupied as well as unoccupied receptors (Pousen et al, 1985).
The human ER is a member of a family of nuclear receptors for small hydrophobic ligands such as thyroid hormone, vitamin D, retinoic acid and the steroid hormones. Each receptor has a ligand-binding domain, a hinge region, a DNA-binding domain and a variable or regulatory domain. The ER gene is located on the long arm of chromosome 6 (q24-27) and comprises eight exons and intervening introns spanning at least 140 kilobases. Binding of the ligand to the receptor is thought to result in an allosteric alteration that allows the hormone-receptor complex to bind to its DNA response element in the promoter region of a target gene. In the absence of hormone binding, the domain appears to be inhibitory in function, preventing transcriptional activation. Besides establishing the allosteric association of the hormone binding and the regulatory domains, the sequences contained in the hinge region are critical in directing the ER and progesterone (PR) proteins to the nucleus after they are synthesized in the cytoplasm. The DNA-binding domain has many basic amino acids, some of which are repeating units folded into a ''fingered" structure coordinated by a zinc ion, known as the "zinc finger" (Schwabe et al, 1990). The ER and PR appear to enhance the transcriptional activity of selected genes. The actual mechanism is not known but probably involves interactions between receptors and other transcriptional factors with the promoter regions of the respective genes. In the current model of ER action, estradiol diffuses into the cell and binds to the receptor, leading to its dimerization and tight binding to its specific DNA target. Following this binding to the estrogen response element in target genes, there is stimulation to increase the transcription of target genes, some directly or indirectly leading to the establishment of both autocrine and paracrine growth stimulatory loops (Leong & Lee, 1995).
Early studies employing monoclonal antibodies to estrophilin reported both cytoplasmic and nuclear staining, the former being stronger. However, subsequent studies have established an exclusive nuclear localization for ER proteins in both human breast carcinomas as well as other steroid-responsive tissues. This has been confirmed by autoradiographic studies and immunoelectron microscopy has shown the ER protein to be present in the euchromatin portion of the nucleus in breast, endometrial and ovarian cancers as well as in benign endometrium. The cytoplasm in all cases did not reveal presence of the receptor although there was some reaction product in the ribosomes. This latter reactivity was considered to be non-specific although the possibility of synthesis of ER at the ribosomal level was not completely ruled out. Studies demonstrating cytoplasmic localization of ER protein in addition to nuclear localization have mostly employed fluoresceintagged estrogen analogs, whereas modern immunoenzyme techniques utilizing monoclonal antibodies to ER protein have shown only nuclear reactivity.
Applications
Over 100 years ago it was recognized that oophorectomy was associated with clinical remission in women with metastatic breast cancer. Despite the usefulness of hormonal manipulation in some women, only approximately 30% of unselected women with metastatic breast cancer responded to such treatment. There was therefore a need to distinguish those women whose breast cancers are hormone dependent from those whose tumors are hormone independent. Employing cytosol-based ligand-binding assays, it was shown that about 50-60% of women with ER-rich breast cancers responded to hormone treatment, while less than 10% with ER-poor tumors showed a similar response. The relevance of ER status and hormonal treatment in node negative tumors, however, is less clear. It is also clinically recognized that a small proportion of patients whose tumors are receptor negative by cytosol-based assays will show a positive response to hormone treatment and as many as one-third of those with ER-positive tumors may fail to respond to such treatment. On the basis of comparative immunohistologic studies, it is believed that some of these discrepancies are caused by inherent errors of the biochemical method which assays homogenized tissue samples with resultant errors introduced by the inclusion of benign epithelium, the dilutional effect of abundant stroma and inadequate tumor sampling. Indeed, there is recent persuasive evidence, based on hormonal response as the ultimate yardstick, that immunostaining in frozen or paraffin sections is the more accurate measurement of ER status (Pertschuk et al, 1996) and this and many other advantages make immunostaining the "gold standard" to replace cytosol assays (Taylor, 1997).
It has been suggested that ER may be used to identify metastatic breast carcinoma but a variety of other lesions with epithelioid features may also express ER.
These include epithelioid smooth muscle tumors, malignant melanoma, meningioma, sclerosing hemangioma (Leong et al, 1997), desmoid tumors, thyroid neoplasms and cervical, endometrial and ovarian cancers, rendering the marker less useful as a diagnostic discriminant.
Comments
Since the development of clone 1D5, it has become possible to perform immunostaining for ER in routinely fixed and processed tissues inexpensively, accurately and consistently. Some form of HIER procedure is essential when using clone 1D5; in contrast, H222 fails to stain following HIER, indicating that only one epitope of the ER, and not the entire antigen, is retrieved by the heating process. For this reason, it has been suggested that the procedure be called heat-induced epitope retrieval rather than the original term "heat-induced antigen retrieval". It is possible to obtain consistent staining of ER in cytologic preparations by employing clone 1D5 with HIER on smears which are initially completely air-dried before fixation in 10% buffered formalin.
It is important, as with most other antibodies employed for immunohistology, that each laboratory determines its optimal time for epitope retrieval and does not purely rely on the procedures developed for other laboratories. determines its optimal time for epitope retrieval and does not purely rely on the procedures developed for other laboratories.
A concordance of 77-100% exists between immunostaining in paraffin and frozen sections with the dextran-coated charcoal assay (DCC) and we have adopted 10% staining of tumors cells as the cut-off value as it corresponded with 10 fmol/mg of proteins by DCC assay. There is strong correlation-of the percentage of positive cells with the intensity of staining. The results of immunostaining may be expressed subjectively as positive or negative, as a percentage of positive tumor cells, or as a score derived by adding grades of staining (1, 2 and 3, corresponding to mild, intermediate and strong staining) and the percentage as scores of 0, 1, 2, 3 and 4 (corresponding to 10%, 11-25%, 26-50%, 51-75% and > 75%). Computerized image analysis has been claimed to produce an increasing specificity and sensitivity relative to biochemical assays but other studies have shown identical results by image analysis and visual examination and significantly similar agreement between the two and biochemical values.
References
•Balaton AJ, Mathieu M-C, Le Doussal V 1996 Optimization of heat-induced epitope retrieval for estrogen receptor determination by immunohistochemistry on paraffin sections. Results of a multicentric comparative study. Applied Immunohistochemistry 4: 259-263.
•El-Badawy N, Cohen C, De Rose PB, Sgoutas D 1991 Immunohistochemical estrogen receptor assay: quantitation by image analysis. Modern Pathology 4: 30-309.
•Greene GL, Fitch FW, Jensen EV 1980 Monoclonal antibodies to estrophilin: probe for the study of estrogen receptors. Proceedings of the National Academy of Sciences USA 77: 157-161.
•Leong AS-Y, Lee AKC 1995 Biological indices in the assessment of breast cancer. Molecular Pathology 48: M221-M238.
•Leong AS-Y, Milios J 1993 Comparison of antibodies to estrogen and progesterone receptors and the influence of microwave-antigen retrieval. Applied Immunohistochemistry 1: 282-288.
•Leong AS-Y, Chan KW, Leong FJ 1997 Sclerosing hemangioma. In: Corrin B (ed) Pathology of lung tumors. London: Churchill Livingstone, pp 175-188.
•Pertschuk L, Feldman J, Kim Y-D et al 1996 Estrogen receptor (ER) immunocytochemistry in paraffin with ER 1D5 predicts breast cancer endocrine response more accurately than H222Sp in frozen sections or cytosol-based ligand binding assays. Cancer 77: 2541-2549.
•Pousen HS, Ozello L, King WJ, Greene GL 1995 The use of monoclonal antibodies to estrogen receptors (ER) for immunoperoxidase detection of ER in paraffin sections of human breast cancer tissue. Journal of Histochemistry and Cytochemistry 33: 87-92.
•Raymond WA, Leong AS-Y 1988 An evaluation of potentially suitable fixatives for immunoperoxidase staining of estrogen receptors in imprints and frozen sections of breast carcinoma. Pathology 20: 320-325.
•Raymond WA, Leong AS-Y 1990 Estrogen receptor staining of paraffin-embedded breast carcinomas following short fixation in formalin: a comparison with cytosolic and frozen section receptor analyses. Journal of Pathology 160: 295-303.
•Schwabe JWR, Newhause D, Rhodes D 1990 Solution structure of the DNA-binding domain of estrogen receptor. Nature 348: 458-461.
•Taylor CR 1997 Paraffin section immunocytochemistry for estrogen receptor. The time has come. Journal of Histotechnology 20: 97-100.
Bibliografía
Manual of diagnostic antibodies for immunohistology / Anthony S.-Y. Leong, Kumarasen Cooper, F. Joel W.-M. Leong.
