An analysis of microvessel density, androgen receptor, p53 and HER-2/neu expression and Gleason score in prostate cancer . preliminary results and therapeutic implications.

OBJECTIVES: To investigate relationships between microvessel density (MVD), androgen receptors (AR), mutant p53 and HER-2/neu expression and Gleason score (GS) to further understand the tumor biology of prostate cancer (CAP). METHODS: Slides of CAP from patients who underwent radical prostatectomy or channel transurethral resection of the prostate (TURP) were tested for androgen receptors by immunocytochemical assay and MVD was analyzed by staining with antibodies to the endothelial cell membrane molecule PECAM-1/CD-31. The p53 monoclonal antibody D07 and HER-2 9G6 mouse monoclonal antibody were used to assess p53 and HER-2/neu expression, respectively. The results were correlated with GS and clinical stage by multivariate analysis. RESULTS: We found a fourfold greater expression of MVD in prostate cancer specimens compared to neighboring normal prostate tissue. We observed a greater concentration of MVD in the higher Gleason scores (r = 0.40, p = 0. 06), and a correlation of Gleason score with mutant p53 expression (r = 0.57, p <0.05). We did not observe any associations between AR or HER-2/neu to Gleason score. More than half of the patients with specimens with 50% or greater expression of mutant p53 were in stage D2 (T4NxM1b) at the time of biopsy. CONCLUSIONS: We observed a correlation between mutant p53 and GS, and a greater concentration of MVD in the higher GS. Since the neovascularity of prostate tumors can be attenuated by radiation and hormones, while mutant p53 may confer resistance to such treatment, it appears that p53 expression may also play an important role in addition to angiogenesis in the virulence of prostate cancer. These data may aid in allocating patients to different treatment modalities.

Image analysis of androgen receptor immunostaining in prostate cancer accurately predicts response to hormonal therapy.

PURPOSE: Immunostaining for androgen receptor in prostate tumor specimens has revealed that the majority of primary and advanced stage cancers are positive for this regulatory transcription factor. Consequently, its use as a marker for tumor behavior and therapeutic response has been discounted. However, past reports have noted significant heterogeneity of androgen receptor immunostaining between prostate tumor cells in contrast to staining homogeneity in normal epithelium, which indicates that variability in androgen receptor content may exist within certain tumor specimens. To analyze this phenomenon more thoroughly and to determine whether this variability possesses clinical correlates, androgen receptor immunostaining profiles within androgen receptor positive prostate tumor specimens were categorized using an image analysis based system. MATERIALS AND METHODS: Tumor specimens were obtained before hormone therapy from 44 patients with advanced stage prostate cancer and 4 with early stage disease who later had progression. Response to antiandrogen therapy and survival was monitored. Paraffin embedded tumor sections were processed for immunocytochemistry and stained for androgen receptor. A Quantimet image analysis system was used to analyze nuclear immunostaining for androgen receptor and Receptogram patterns were established for each specimen based on univariate distributions of nuclear receptor content and concentration. RESULTS: Data revealed that 17 of 18 responders to hormone therapy possessed type 1 (15) or type 3 (2) Receptograms, which are characterized by a unimodal peak or multimodal peaks within a narrow concentration range. Of the 17 cases that stabilized following therapy 16 had type 3 Receptograms and 1 was characterized as type 1. In contrast, all 13 patients in whom endocrine treatment failed had either type 2 or 4 Receptograms, which are characterized by a highly skewed or bimodal androgen receptor distribution. Positive and negative predictive values for this assay were 100 and 93%, respectively. In addition, the type 1/3 Receptogram patterns were correlated with longer mean survival. CONCLUSIONS: Image analysis of prostate cancer androgen receptor immunostaining with a pattern oriented approach for response is capable of accurately predicting response to hormone therapy in patients with advanced stage disease. Application of this analytic scheme may assist the clinician with therapeutic management of advanced prostate cancer.

Estrogen receptor immunocytochemistry in endometrial carcinoma: a prognostic marker for survival.

UNLABELLED: Only a few parameters such as tumor grade and stage are of value in prognosticating disease course in endometrial carcinoma. Biochemical steroid hormone receptor assays could also be useful but are difficult to perform and interpret. Immunocytochemical assay (ICA) might be the method of choice for detecting endometrial receptors. METHODS: Frozen tissue from 78 cases of endometrial adenocarcinoma was examined for the presence of estrogen (ER) and progesterone receptors (PgR) with specific monoclonal anti-receptor antibodies and the peroxidase-antiperoxidase method. In over 60 cases, frozen tissue was also assayed for ER and PgR by biochemical means. RESULTS: Fifty-five (71%) of the endometrial carcinomas were ERICA-positive and 55 (71%) PgRICA-positive. Although both ERICA and PgRICA correlated significantly with biochemical ER and PgR only ERICA was predictive of survival. A woman with a negative ERICA was 4 times more likely to die of her disease than if she were ERICA-positive (P = 0.009; mean follow-up, 37.5 months). Three cases ERICA-positive and PgRICA-negative survived while 3 others ERICA-negative and PgRICA-positive died. CONCLUSION: ERICA, a technique easy to perform and interpret at the community hospital level, appears to provide prognostic information independent of tumor stage and grade. Such information might be of value in planning postoperative therapies for women with endometrial cancer.

Estrogen receptor immunocytochemistry in paraffin embedded tissues with ER1D5 predicts breast cancer endocrine response more accurately than H222Sp gamma in frozen sections or cytosol-based ligand-binding assays.

BACKGROUND: Historically, estrogen receptor (ER) determinations have been made by the ligand-binding assay of tumor homogenates, primarily by the dextran-coated charcoal method (DCC). Immunocytochemical assays (ICA) for ER are more recent and have been executed mostly on frozen sections with the monoclonal antibody H222Sp gamma (H222). Lately, new monoclonal antibodies derived by recombinant ER technology have been developed that work well on paraffin embedded, formalin fixed tissue sections. However, there is little information as to whether such assays prognosticate endocrine response. METHODS: Using antigen retrieval, the immunoglobulin G1 monoclonal antibody ER1D5, and the streptavidin-biotin detection system, 74 patients with breast cancer in whom endocrine response was known were assayed and the results compared with ER by DCC and ER by ICA in frozen section with H222. RESULTS: ER1D5 in paraffin provided the highest correlation with endocrine response (Kendall's tau [r] = 0.57; P<0.001) whereas ER by DCC failed to correlate (r= -0.002; P<0.99). ER1D5 in paraffin correlated weakly though significantly with DCC (Kappa Statistic [K] = 0.204; P<0.02). H222 in frozen sections also correlated moderately with endocrine response (r = 0.34; P<0.001). CONCLUSIONS: ER can be detected in routine tissue sections processed with antigen retrieval and ER1D5, and can be relied upon to provide accurate prognostic information regarding response to endocrine therapies in breast cancer patients.

Immunostaining for prostate cancer androgen receptor in paraffin identifies a subset of men with a poor prognosis.

BACKGROUND: Knowledge of androgen receptor (AR) content could help predict hormone response and disease course in prostate cancer. However, determination of AR by biochemical assay is difficult. An immunohistochemical assay (ICA) would solve most difficulties and be especially useful if it could be performed on paraffinized tissue. EXPERIMENTAL DESIGN: AR was studied in paraffin sections from 90 men for whom endocrine response, survival (except one case), and/or biochemical AR was known. After Ag retrieval in a microwave oven, a polyclonal anti-AR Ab was used with the peroxidase antiperoxidase method. Results were semiquantified using a Histoscore (Hscore) and were correlated with biochemistry, endocrine response, and survival. RESULTS: Only 15 patients were AR-negative. AR-ICA did not correlate with biochemistry, Gleason score, stage, or ethnicity but did correlate with endocrine response and survival. The average Histoscore was significantly lower in patients with progressive disease (p < 0.05). In a Cox's regression analysis of survival (mean follow-up = 30 months) AR-ICA was a significant predictor (p = 0.015). Risk of death was 2.5 times greater for a patient with a negative assay compared with one with a positive result. CONCLUSIONS: Our data indicate that AR status by ICA may be a useful predictor of survival and endocrine response in prostate cancer. Further studies are needed to confirm these results because the assay could impact significantly on management.

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.

Verrucous carcinoma of the leg positive for human papillomavirus DNA 11 and 18: a case report.

Human papillomavirus (HPV) types 6 and/or 11 have been associated with benign lesions, while types 16, 18, 31, and 33 are prevalent in malignant lesions. This case report describes the findings in a verrucous carcinoma of the leg, which was examined for HPV types 11, 16, and 18 by in situ DNA hybridization. The lesion gave positive results for HPV subtypes 11 and 18, a combination that, to our knowledge, has not been previously reported in this neoplasm.

Comparison of microscopic imaging strategies for evaluating immunocytochemical (PAP) steroid receptor heterogeneity.

Receptogram analysis was compared with three other imaging strategies for immunocytochemical assay of estrogen receptors. These included nuclear-specific methods for analysis of nuclear integrated optical density (IOD) or mean optical density (MOD) histograms, and field-specific methods, where the pixel optical density (POD) histogram was evaluated for the composite nuclear phase. Measurements in culture and in breast cancer cryosections were treated separately to isolate geometric considerations. In culture receptograms the modality of IOD and MOD histograms and their bivariate contour maps revealed one, two, or more subpopulations with discrete receptor content and concentration. However, when the field of nuclei was imaged as a whole, regardless of the number of subpopulations, POD histograms showed two minima, defining three intranuclear phases. This was due to mottling and variegation of intranuclear chromatin and nucleolar immunostaining and not to differences between nuclei. These limitations were also revealed in breast cancer sections. In POD histograms, % unstained pixels did not provide a reliable estimate of % receptor negative nuclei, as determined by their enumeration. In sections, correction of IOD for nuclear volume variability was essential to suppress artifactual peaks not representing differences in receptor content. This was achieved by multiplying nuclear IOD by the spherical nuclear radius (S) of individual slab sections. Peaks of IOD(S) then reflected receptor content on a true ratio scale. Only receptogram analysis, which incorporates these strategies, permitted objective evaluation of receptor heterogeneity at the level of tumor subpopulations.

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.

Quantitative imaging of immunocytochemical (PAP) estrogen receptor staining patterns in breast cancer sections.

"Receptogram Analysis" has been developed as a pattern-oriented approach for predicting endocrine response in breast cancer based upon quantification of the estrogen receptor immunocytochemical assay (ERICA), using a Quantimet Imaging System. Response prediction was evaluated in 58 stage III and IV patients receiving endocrine therapy (primarily Tamoxifen). The Receptogram is a composite of the univariate distributions of nuclear receptor content, IOD(S), and concentration (MOD), and their bivariate contour plot; where (S) is the calculated nuclear radius in section. MOD distributions were classified into four types based upon peak modality and kurtosis (I-IV), and contour plots were classified into four subtypes (A-D) based upon contour slope. Patients failing therapy were ERICA--or their receptogram revealed co-existent ER+ and ER- tumor cells (type II), highly skewed MOD distributions lacking defined peaks (type IV), or contours with nearly horizontal slope (type C). Response was realized in 9/16 type I patients, with a single positive MOD peak, and in 9/15 type III patients, with discrete, multimodal MOD peaks. In contrast, 0/8 type II, 0/12 type IV, and 0/10 type C patients were responders. Receptogram analysis was superior to cytosol assay (DCC) as a response discriminant: positive predictive value, 53% vs. 33%; negative predictive value, 100% vs. 75%; sensitivity, 100% vs. 83%; specificity, 68% vs. 23%; and accuracy, 78% vs. 41%, respectively. Alternately, patients were assigned to potentially responsive or non-responsive groups based upon thresholded mean receptor parameters: field MOD, mean nuclear MOD (NMOD), and mean NMOD(PF) where PF is the ER+ nuclear fraction. While these parameters correlated with DCC (r = .72, 0.69, and 0.69), they were only marginally better in predictive value.

Immunocytochemical assay of progesterone receptors in paraffin-embedded specimens of endometrial carcinoma and hyperplasia: a preliminary evaluation.

An immunohistochemical method for the detection of progesterone receptors (PgR) using the monoclonal anti-PgR antibody KD 68 was utilized to study paraffin-embedded tissue sections from women with endometrial carcinoma and hyperplasia. Stromal as well as myometrial nuclear PgR were nearly always apparent. In carcinoma, 11/24 (46%) of cases showed epithelial positivity, whereas in hyperplasia 8/9 (89%) were PgR-positive (P less than 0.05). Initial biochemical PgR assays by the dextran-coated charcoal method were compared with results of PgR-immunocytochemical assays (ICA) in the paraffin-embedded tissue and were in concordance in 92%. In the one discordant specimen, PgR-ICA-negative tumor cells were seen infiltrating PgR-ICA-positive myometrium, and the biochemical assay was thus felt to be falsely positive. Twelve additional cases of endometrial carcinoma were also studied for estrogen receptor (ER) by immunocytochemistry. Two were positive for both ER and PgR, while five were negative for both receptors. The immunocytochemical methods described allow for analysis of routinely fixed, paraffin-embedded specimens, thus permitting analysis of very small specimens and archival material.

Progesterone receptors in the human heart and great vessels.

Progesterone receptors (PgR) were identified in 31 of 50 specimens of human (men and women) thoracic ascending aorta, internal carotid, coronary artery, and left atrial appendage. This was accomplished with a peroxidase-antiperoxidase immunocytochemical assay employing a highly specific monoclonal antibody to primate PgR. In the aorta, specific staining was seen in the nuclei of smooth muscle cells and endothelium of intima, media, and adventitia. In the myocardium, staining was localized to the nuclei of the myocardial fibers. In internal carotid and coronary arteries, PgR was localized to endothelial nuclei of intima, and in vascular channels within the atherosclerotic plaques. PgR was also visible in the smooth muscle cell nuclei of uninvolved media and intima and at the plaque periphery. In contrast, receptor was not identified in vessels of the human uterus, breast, prostate, kidney, or gastrointestinal tract. These findings suggest that the heart and great vessels are target organs for steroid hormones.

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.

Quantitation of the immunocytochemical assay for estrogen receptor protein (ER-ICA) in human breast cancer by television imaging.

A Quantimet 720D Image Analysis System has been programmed for light microscopic evaluation of the nuclear estrogen receptor distribution in frozen sections of human breast cancer stained by the peroxidase-antiperoxidase method using monoclonal antibodies to estrogen receptor protein (ER). This method provides precise criteria for distinguishing ER-positive and -negative cells and a sensitive and reproducible means for densitometric quantification of the staining patterns. Although imaging sequence and graphic analysis are automated by computer programs, light pen interaction provides supervision of feature selection. Imaging of the immunocytochemical assay (ER-ICA) in 50 patients revealed marked heterogeneity of nuclear estrogen receptor concentration varying over a nearly 100 fold concentration range. Various ER concentration patterns were evident: (I) distributions with a single peak (CV = 5%) present at various concentration levels; (II) bimodal distributions, revealing co-existent ER-positive and ER-negative subpopulations; (III) multimodal distributions with a number of resolvable concentration peaks; and (IV) highly skewed distributions with or without discernible peaks, frequently extending over the entire concentration range. Statistical methods of de-convolution were applied to determine the frequency and ER concentration characteristics of component subpopulations in the mosaic cases and for resolving the proportion of ER-positive and -negative cells. An approach for evaluating nuclear ER content in conjunction with ER concentration patterns in individual patients revealed whether spread in the ER concentration distribution resulted from differences in nuclear ER content or from variability in nuclear volume distribution.

Immunocytochemical assay of estrogen receptors in endometrial carcinoma with monoclonal antibodies. Comparison with biochemical assay.

Specimens of endometrial adenocarcinoma, surgically obtained from 18 women, were analyzed for distribution of estrogen receptors by an immunocytochemical assay, employing monoclonal anti-estrophilin antibodies and the peroxidase-antiperoxidase technique. Results were compared with biochemical receptor analyses, and were in concordance in 83% of them. Marked tumor cell and tissue receptor heterogeneity were apparent with the immunocytochemical method, and a variety of patterns of nuclear staining in positive tissue samples were revealed. These results indicate that the immunohistologic method will provide a number of entirely new variables that may eventually be correlated with both clinical and pathologic features of this malignancy, and may prove to be of value in the prediction of clinical endocrine response.

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 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.