Immunocytochemical analysis of estrogen receptors as a predictor of prognosis in breast cancer patients: comparison with quantitative biochemical methods.

Biochemical quantitation of estrogen receptors has been used to predict prognosis in breast cancer. Immunocytochemical analysis of estrogen receptors correlates with biochemical analysis but has very few follow-up studies in the literature to validate it as a prognostic indicator. 257 patients were followed for up to 10 years (median, 6.2 years) after primary surgical treatment. Estrogen receptor analysis using both biochemical and immunocytochemical techniques was performed on their tumor specimens. Patients with positive estrogen receptor values had longer survival than patients with negative values. This was demonstrated by both methods in the first 5 years of follow-up but only by immunochemistry after 5 years. The relationship between estrogen receptor status and disease-free interval was less strong than with survival. This study demonstrates that immunocytochemical estrogen receptor analysis was of prognostic significance.

Detection of estrophilin in frozen sections of breast cancers using an estrogen receptor immunocytochemical assay.

Estrogen receptor (ER) was detected in frozen sections of 36 breast carcinomas using an antiestrophilin monoclonal antibody according to an immunocytochemical technique elaborated and made available by Abbott Laboratories in the form of a kit (ER-immunocytochemical assay monoclonal). Immunostaining was confined to the nuclei of the carcinoma cells. In all positive specimens, nuclei with different staining intensities were present in addition to a variable number of unstained nuclei, presumably because of functional heterogeneity. Of the 36 carcinomas, 27 displayed positive immunostaining, 4 had no staining, and in 5 the staining was borderline. All specimens were assayed for ER content by the dextran-coated charcoal (DCC) technique. When the DCC values were compared with the results of immunostaining it was found that 4 tumors were negative and 27 were positive by both techniques, whereas of 5 cases with borderline staining 3 were negative by DCC and 2 had low DCC values. These correlations proved to be highly significant (P much less than 0.001). The number of stained nuclei (extent of staining) related to the DCC status in a significant manner (P less than 0.01), whereas the intensity of staining did not (P greater than 0.10). These results indicate that immunocytochemical visualization of ER using Abbott's "ER-Immunocytochemical Assay Monoclonal" kit is an easy, reproducible, and reliable technique.

Use of a monoclonal anti-estrogen receptor antibody in the immunohistochemical evaluation of human tumors.

A monoclonal antibody to human estrogen receptor protein (H222 Sp gamma), amplified via immunoperoxidase techniques, was used in the analysis of estrogen receptor in 452 breast carcinomas, 100 endometrial carcinomas, and 15 melanomas. Immunohistochemical evaluation incorporated both intensity and distribution of staining (HSCORE). Quantitative estrogen receptor content was determined by dextran-coated charcoal analysis and sucrose density gradient analysis. In all cases H222 Sp gamma localized in the nucleus of target cells. A semiquantitative correlation existed between HSCORE and biochemical assays for breast and endometrial tissues. The sensitivities and specificities for HSCORE as compared to the biochemical assays ranged from 80 to 95% and from 74 to 94%, respectively. HSCORE correlated with tumor grade for breast and endometrial carcinoma. Immunohistochemical evaluation showed no specific staining in melanomas. The data suggest that immunohistochemical receptor localization provides information complementary to standard biochemical assays in the tissues studied.

Absence of estrogen receptor in human melanoma as evaluated by a monoclonal antiestrogen receptor antibody.

Controversy regarding the presence of estrogen receptor proteins in human melanomas persists despite extensive investigations on this subject. While apparent high-affinity binding has been observed using dextran-coated charcoal assays, several other characteristics of receptor protein have not been observed. The production of free water on incubation of tritiated estradiol (labeled in the C2 position) with melanoma cytosols suggests the possibility that the apparent binding observed is due to phenomena other than specific receptor-steroid interactions. Melanomas from 15 patients were evaluated for the presence of estrogen receptor using immunocytochemical techniques with a monoclonal antibody directed against the human estrogen receptor protein (H222 Sp gamma). Immunohistochemical evaluation included intensity and distribution of staining. None of the 15 cases demonstrated specific immunohistologic reactivity with the anti-receptor antibody. Control breast and uterine tissue confirmed the specificity and sensitivity of the methods. These results suggest that the apparent estrogen-binding capacity of human melanoma tissues is the result of interactions other than with estrogen receptor, and reaffirm the need to investigate alternate steroid protein interactions, such as catechol estrogen formation, in studying sex steroid influences on human melanoma.

Estrogen receptor analyses. Correlation of biochemical and immunohistochemical methods using monoclonal antireceptor antibodies.

Attempts at histochemical localization of estrogen receptor with anti-steroid antibody or some fluoresceinated estrogens have given unacceptable sensitivities and specificities when compared with biochemical methods or clinical response. In the present study a monoclonal antibody against estrogen receptor (H222 Sp gamma) was used on cryostat sections of freshly frozen breast tumors with a peroxidase-antiperoxidase immunoperoxidase technique. Biochemical receptor analyses were by dextran-coated charcoal analyses. Tumors from three separate cohorts of patients were studied as follows: population A, 62 primary breast cancers from 1983; population B, 72 primary lesions stored from 1976 to 1983; and population C, 23 patients with metastases, treated with hormonal therapy. Distinct staining was seen in the cell nucleus. A semiquantitative relationship was seen between histochemical score assessment of staining and biochemical assay in each cohort. The sensitivity and specificity using a threshold of 75 for the histochemical score and more than 20 femtomoles/mg of protein for dextran-coated charcoal analyses were as follows: population A, specificity, 89%, and sensitivity, 95%; population B, specificity, 94%, and sensitivity 88%; and for population C, the comparison was with objective clinical response yielding specificity, 89%, and sensitivity, 93%.

Immunohistochemical assessment of estrogen and progesterone receptors in stored imprints and cryostat sections of breast carcinomas.

A peroxidase-antiperoxidase technique was used to visualize estrogen and progesterone receptors in stored imprints and cryostat sections of breast carcinomas that were prepared at the time of biopsy or frozen section diagnosis. This was done to provide an alternate technique for the assessment of the receptor status of tumors that could not be adequately assayed with other biochemical or immunocytological methods. Fixation in Zamboni's fixative followed by passage through cold methanol and acetone before storage at -80 C insured good preservation of the receptor proteins over extended periods of time (up to 56 weeks). Immunostaining of these stored preparations with monoclonal antibodies against estrogen receptor (H222) and progesterone receptor (JZB39 and KD68) showed a high degree of correspondence with immunocytochemical assays (ER-ICA and PR-ICA) and biochemical analysis. This technique is easy to perform and provides reliable information, even in tumors that are too small and/or ill defined to permit separate sampling for receptor assays.