Not only the presence, but the amount of estrogen receptor (ER) has been shown to correlate with clinical response to endocrine therapy in breast cancer. A total of 130 breast cancers and 26 prostatic cancers were subjected to steroid binding quantification and semiquantification methods to allow for a comparison between techniques. As a consequence, it may be necessary to develop methods to semiquantify or quantify histochemical techniques for the detection of steroid binding, in order to determine if the amount of ligand which may be bound is of significance. The ER Immunocytochemical Assay of Greene is a method which employs monoclonal antibodies to ER, a bridging antibody, and peroxidase-anti-peroxidase complexes. Several investigators have reported performing competitive binding studies by exposing parallel tissue sections, respectively, to ligand-conjugate and ligand-conjugate plus an excess molar concentration of competitor ligand. One of the virtues of microfluorometry is in assessing competitive binding studies since numerical evidence of inhibition of ligand-conjugate binding can, thus, be obtained.

This article reviews the development of steroid hormone receptor detection using the biochemical approach and the disadvantages inherent in these systems. The early history of methods for the in situ detection of receptors is related, culminating in the development of histologic immunoassays using monoclonal antibodies. Correlation of the latter with disease-free and overall survival and with clinical endocrine response are presented together with preliminary findings utilizing a new generation of antireceptor antibodies. Current problems with test scoring and interpretation are detailed with a section devoted to image analysis for assay quantification. Emphasis is placed on the fact that because so many different antibodies and tissue substrates have been employed in the past, these histologic receptor immunoassays cannot yet be considered to be fully validated. It is stressed that different laboratories must employ the same antibodies, substrates, detection systems and methods of scoring, and then correlate findings with measurable clinical end points. Only then can the assays be included in the brief list of approved breast cancer prognostic and predictive markers to eventually replace the routine biochemical methods of steroid receptor detection. ▪

It is customary to submit only one portion of a breast cancer to determine if there is amplification or overexpression of the proto-oncogene HER-2/neu. In routine studies of the expression of neu in breast cancer, however, we noted discrepancies in intratumoral positivity. To investigate this phenomenon further, multiple tumor specimens (129 samples) from 41 women with breast cancer were examined. Forty cases were analyzed for neu amplification by slot blot assay and 18 with fluorescent in situ hybridization. Neu overexpression was determined using four different specific antibodies. In more than 50% of cases there were discrepancies in results between the tissue blocks examined. This was evident in both inter- and intra-assay comparisons. It is concluded that intratumoral heterogeneity of neu amplification/overexpression in breast cancer exists to a far greater degree than previously recognized and could be a responsible factor for conflicting published data regarding neu's prognostic significance. Examination of only one tumor sample may not give a true indication of either amplification or overexpression of this oncogene.

A significant volume of clinical, biochemical, and autoradiographic data suggest that mammalian arteries possess estrogen receptors. In most cases, experimental evidence was obtained from species other than humans. We employed immunohistochemistry with specific anti-estrogen receptor monoclonal antibodies to localize and map estrogen receptors within the human cardiovascular tree by examining arteries from men and women undergoing a variety of surgical procedures, as well as vessels from cadaveric donors (214 specimens) and from female macaques (84 specimens). In both species, the receptors were primarily limited to nuclei of endothelium and smooth muscle cells in the media of mediastinal vessels and of arteries supplying the female reproductive organs. No receptor was found in myocardium. No relationship was discovered between patient age, sex, and receptor positivity, implying that clinical differences might be a function of circulating hormones rather than differences in location of receptor.

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.

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 dagger 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 I 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.

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.

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 G(I) 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. (C) 1996 American Cancer Society.

BACKGROUNDKnowledge 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 DESIGNAR 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.RESULTSOnly 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.CONCLUSIONSOur 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.

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.

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.

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.

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.

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.