Immunocytochemical assay for androgen receptors in prostate cancer: a prospective study of 63 cases with long-term follow-up.

BACKGROUND: Numerous problems are associated with biochemical androgen receptor (AR) assay performance and interpretation in prostatic cancer. The purpose of this study was to determine if a novel immunocytochemical AR assay performed on intact tissue sections would prove useful in prognosticating endocrine response and survival. METHODS: A prospective study was done on 63 prostatic carcinomas maintained in liquid nitrogen for over a decade. The study used the peroxidase-antiperoxidase system and a polyclonal anti-AR antibody. RESULTS: Marked tissue and cellular heterogeneity of nuclear AR was apparent. A cut-off of 10% AR-positive cells maximized assay prognostic efficiency. Frequency of positivity was 48% and correlated significantly with endocrine response (p = 0.03), time to progression (p = 0.0016), and survival (p = 0.02), but not with grade, stage, or ethnicity. CONCLUSIONS: This AR assay could be prognostically useful in the clinical management of prostate cancer and is suitable for use in the community hospital laboratory.

Estrogen receptor immunocytochemistry: the promise and the perils.

Estrogen receptor immunocytochemistry (ERICA) is favored over dextran-coated charcoal (DCC) or sucrose gradient assay (SGA) by many pathologists and oncologists since it allows an estimation of tumor cell and tissue heterogeneity and permits assays to be performed on specimens not suitable for DCC/SGA. Additionally, ERICA can be performed with greater ease and with less expense at the level of the community hospital pathology laboratory. Initially, like DCC/SGA, ERICA had to be done on fresh or frozen tumor samples or face a significant loss in sensitivity when applied to formalin-fixed, paraffin-embedded sections. Recently, several anti-estrogen receptor (ER) antibodies have appeared which can be successfully employed to assay routinely prepared tissue sections if used in conjunction with new antigen-retrieval techniques such as the microwave oven and citrate buffers. However, more work is needed to correlate results of these new procedures with biochemical ER assays, endocrine response, and survival before they can be reliably employed as prognostic parameters. Furthermore, if any ER assay is to be useful and valid, strict attention must be paid to details of specimen collection, freezing, and fixation in order to inhibit receptor degradation and false negative results.

Steroid hormone receptor immunohistochemistry and amplification of c-myc protooncogene. Relationship to disease-free survival in breast cancer.

BACKGROUND: It is important to develop parameters that aid in prognosticating which patients with breast cancer are more likely to have a rapid disease course and therefore might benefit from early aggressive therapies. METHODS: Specimens from two groups of women, deliberately selected because their clinical courses differed greatly, were studied to detect amplification of the protooncogenes c-myc, int-2, and C-erbB-2/neu by slot-blot assay, the estrogen receptor (ER), and the progesterone receptor (PR) by both biochemical and immunohistochemical procedures (ERICA and PRICA). One group of 50 patients had a prolonged disease-free interval after initial surgery (mean, 6.4 years); the other group of 52 women had had rapid disease recurrence (mean, 1.4 years) or progression (5 patients died of disease within 1 year of diagnosis). The patients were selected from 1700 consecutively accessioned cases if they fit the study criteria and sufficient tissue was available for oncogene hybridization studies. RESULTS: The two groups differed statistically by stage, number of involved axillary lymph nodes, ERICA and PRICA results (P = 0.001), and amplification of c-myc (P = 0.003). The percentage of patients with rapid disease recurrence and progression increased from 0-93% when risk factors changed from best case (ERICA and PRICA results, positive; c-myc, not amplified; and axillary nodes, not involved) to worst case (ERICA and PRICA findings, negative; c-myc, amplified; and axillary nodes, involved). CONCLUSIONS: Women with these worst-case parameters were more likely to have a recurrence sooner and rapidly progressive disease. They might benefit from early aggressive therapeutic measures.

Immunocytochemical estrogen and progestin receptor assays in breast cancer with monoclonal antibodies. Histopathologic, demographic, and biochemical correlations and relationship to endocrine response and survival.

Breast cancer specimens from 600 women were assayed for estrogen receptors (ER) using an immunocytochemical assay (ICA) employing the monoclonal antiestrophilin antibody H222 Sp gamma. Results showed significant correlation with biochemical ER determinations as well as with tumor grade and menopausal status. In 449 cases, results of progesterone receptor assay by ICA using the monoclonal anti-PgR antibody KD 68, also correlated significantly with biochemical PgR measurements. The ERICA/PgRICA positivity was significantly more frequent in postmenopausal white women. Colloid carcinomas were most likely to be ERICA positive and PgRICA positive whereas medullary carcinomas were most often negative. In 47 patients with advanced mammary carcinoma, results of ERICA and PgRICA were more closely related to endocrine response than those of ER and PgR by dextran-coated charcoal assay (DCC). In 339 women with Stage I or Stage II breast cancer, ERICA was significantly associated with disease-free survival. Analysis by Cox's proportional hazard model, however, showed PgRICA to be the best predictor of survival and disease-free survival in 197 women at the same stages of disease. These data indicate that ICA is more predictive of prognosis than biochemical ER and PgR. The ease of ICA performance coupled with these results indicate that the method is an acceptable substitute for DCC in analyzing breast cancers for ER/PgR.

Immunocytochemical detection of progesterone receptor in breast cancer with monoclonal antibody. Relation to biochemical assay, disease-free survival, and clinical endocrine response.

A new immunocytochemical assay for progesterone receptor (PgR-ICA) employing the monoclonal antibody JZB 39 was used to study tumors from two series of patients with breast cancer. In Series 1 assay results were in agreement with those of biochemistry in 76% of 338 cases (P less than 0.001) and in 54% of 101 cases in Series 2 (P less than 0.001). Agreement was better in Series 1 because it included fresher, previously untouched specimens. There were 70 patients in Series 1 with known clinical endocrine response. A negative assay correlated with disease progression in 45 of 57 patients, significantly better than with biochemistry (P = 0.013). In comparing 39 women with rapid disease progression with 39 free of disease at 5.1 years, those with PgR-ICA-positive tumors were over four times more likely to remain disease-free than those with negative results (P = 0.007). Product moment life-table analysis of 79 patients from Series 2 showed a significantly better cumulative survival for those with PgR-ICA-positive tumors (P = 0.047). These findings indicate that PgR-ICA should be of value in planning therapy and predicting disease course in breast cancer patients.

Histochemical estrogen binding. An independent predictor of recurrence and survival in stage II breast cancer.

Cox's proportional hazards regression model was used to analyze the prognostic significance of multiple variables affecting recurrence and survival in patients with Stage II breast cancer. Among the variables were biochemical estrogen (ER) and progesterone receptor (PgR) values and results of a histochemical estrogen-binding assay using a fluoresceinated bovine serum albumin-estradiol conjugate where carrier and label were bound at position 17. In 190 cases ER and PgR were not found to be significantly associated with either disease recurrence or patient survival. On the other hand, patients with tumors that were demonstrably "rich" in estradiol ligand conjugate binding by histochemistry experienced both a longer disease-free interval (P less than 0.03) and survival (P less than 0.02) than did patients whose tumors were "poor" in conjugate binding or showed a heterogeneous population of positively and negatively stained cells. A patient with a tumor rich in estrogen binding was five times more likely to survive than a patient with a neoplasm that was poor in estrogen binding by histochemistry. These results indicate that the histochemical technique used provides new and independent parameters for determination of prognosis in Stage II breast cancer.

Immunohistologic localization of estrogen receptors in breast cancer with monoclonal antibodies. Correlation with biochemistry and clinical endocrine response.

Breast cancer specimens from 114 patients were assayed for the presence of estrogen receptors (ER) utilizing highly specific, monoclonal antiestrophilin antibodies and the peroxidase-antiperoxidase technique. Results were compared with conventional ER determinations by the dextran-coated charcoal method (DCC) and were in agreement as to positivity and negativity in 86%. Semiquantified immunocytologic assay results were in accord with the level of ER as measured by DCC in 66%. The tumors studied included 43 from patients with Stage IV disease where clinical response to hormonal manipulation was known. In the latter group, the immunohistologic method had a sensitivity similar to that of DCC but showed a superior positive predictive value and a significantly better specificity. These results indicate that this new method is a valuable laboratory tool, enabling prediction of hormone responsiveness in advanced mammary carcinoma and capable of performance at the community hospital level.

Heterogeneity of estrogen binding sites in breast cancer: morphologic demonstration and relationship to endocrine response.

Breast cancer specimens from 184 patients were analyzed for estrogen binding by two different histochemical techniques using conjugates of estradiol, bovine serum albumin, and fluorescein. In one conjugate estradiol was bound at position 6, in the other at position 17. Results were in agreement in 64% (p less than .001), but obvious differences in ligand distribution were noted. Results were also correlated with estrogen receptor (ER) analysis by dextran-coated charcoal assay (DCC) and were in accord in 65% and 67% of specimens respectively (p less than .001). In 114 cases, the tissue samples were also studied with the estrogen receptor immunocytochemical assay (ERICA) of Greene and his colleagues, which employs monoclonal antibodies to ER protein. Results were in accord with DCC in 86% (p less than .001). The pattern of staining with ERICA differed from that of either histochemical method. In 43 cases assay results were correlated with clinical endocrine response. Overall, the best statistical prognostic parameters were obtained with ERICA. Analysis of combined assay results revealed that patients with assays positive by all techniques were the most likely to respond to hormonal treatment (p less than .001), whereas if one or more assays were negative the chances for a good response were significantly less favorable. These data suggest that DCC and ERICA are both a measure of the same estrogen binding site (type I) while the histochemical methods apparently identify two other separate and distinct sites (putative type II sites). A degree of positive interaction may exist between these multiple estrogen binding sites.

Heterogeneity of steroid binding sites in prostatic carcinoma: morphological demonstration and clinical implications.

Prostatic neoplasms were studied for estrogen binding using four methods. Two employed fluorescent estrogen histochemical ligands, one was a new immunocytochemical technique using specific monoclonal antibodies to human estrophilin, and the last procedure was conventional biochemical dextran-coated charcoal assay. Results indicated that the fluorescent ligands recognized closely associated but separate estrogen-binding sites (putative type II sites) which in turn differed from the binding site measured biochemically. Studies with the monoclonal antibodies were nearly always negative, suggesting that prostatic estrogen receptor might vary antigenically from that present in breast and endometrium. Histochemical and biochemical androgen-binding studies were also compared and showed a close association. In the prediction of hormonal response in advanced prostate cancer both showed high sensitivity and low specificity. The addition of estrogen-binding data did not improve the predictive value of the androgen-binding histochemical assay. However, combining results of the biochemical and histochemical androgen-binding assays resulted in significant improvement of the specificity without loss of sensitivity, suggesting that there is a degree of positive interaction between the binding sites assayed by the two methods.

Correlation of histochemical and biochemical analyses of androgen binding in prostatic cancer: relation to therapeutic response.

A histochemical technique for the detection of androgen binding in prostatic cancer was performed on specimens from 108 patients and compared with a biochemical method in a double blind study of 77. Statistical analyses showed a significant agreement between the two assay systems for the qualitative and quantitative presence or absence of specific androgen binding, as well as for the subcellular localization of binding in nucleus and/or cytoplasm. Although the number of cases studied was too small for statistical analysis, there appeared to be good correlation between histochemical androgen binding results and clinical response, or lack of response to hormonal manipulation in 20 patients with State C and Stage D carcinoma. No correlation was evident between androgen binding and tumor grade or clinicopathologic stage of disease of either histochemistry or biochemistry.

Immunohistologic and histochemical methods for detection of steroid binding in breast cancer: a reappraisal.

A review of the current literature on immunohistologic and histochemical methods for the detection of steroid hormone binding sites in breast cancer, reveals that many, but not all of the criteria for establishing hormone-receptor binding interactions have been met. These include tissue specificity, binding between labeled ligands and soluble receptor in vitro, correlations between histochemical and biochemical assays, as well as between histologic procedures and tumor hormone responsiveness. However, histochemical binding phenomena do not appear to follow classical receptor dogma in regard to the concentration of ligand required, or specificity of binding as determined by competitive binding assays. It is concluded that these histologic techniques may be detecting classical receptor that may be reacting differently than would be expected from biochemical analyses, Types II and III binding sites, and/or organelle and membrane-bound receptors. Certainly no current method should presently be promoted as a laboratory method for the detection of classical receptor. New immunocytologic procedures employing specific, antireceptor sera currently under development, may obviate many of the criticisms leveled against earlier methods.

Histochemical analyses of steroid hormone receptors in breast and prostatic carcinoma.

Histochemical analyses estrogen (ER) and progesterone (PgR) receptors in breast cancer were statistically correlated with results of dextran-coated charcoal (DDC) and sucrose gradient assays. Correlated for ER was 91% of 363 cases, and for PgR 88% of 255 specimens. Breast cancer ER/PgR positivity by histochemistry correlated with a favorable clinical response to endocrine therapies in 72% of 25 cases, while ER/PgR negativity correlated with a lack of response in 96% of 22 cases with Stage IV disease. Nuclear ER/PgR correlated with a poor response to therapy in 8 of 12 patients. An in vitro technique to detect nuclear translocation of ER revealed two groups of ER positive cases, with 11 of 17 exhibiting translocation and 6 not displaying translocation. In prostatic carcinoma, 72% of 65 men were positive for ER and/or androgen receptor. Comparison of specimens obtained without and with electrocautery revealed a preponderance of nuclear binding in the latter, suggesting heat-induced nuclear translocation of receptor. coumestrol, a naturally fluorescent, entirely unaltered estrogen was also used for histochemical detection of ER. Results correlated with ER by DCC in 87% of 61 breast cancers. Coumestrol was additionally used to visually observe receptor and nuclear translocation of ER in intact whole cells in culture.