Conjugation of interferon alpha to a humanized monoclonal antibody (HuBrE-3vl) enhances the selective localization and antitumor effects of interferon in breast cancer xenografts.

Human mammary carcinoma xenografts (MCF-7) growing in nude mice were treated with natural interferon alpha (n-IFN-alpha) alone or conjugated to a humanized monoclonal antibody (MoAb) anti-breast mucin (HuBrE-3vl) or to irrelevant human IgG1kappa. The IFN and the conjugates were administered as 20 intra-lesional (i.l.) injections to 1 of 2 xenografts in each mouse, or i.p. The growth inhibitory effects of HuBrE-3vl/nIFN-alpha were significantly greater than those of nIFN-alpha used as a single agent or conjugated to HuIgG1kappa. These effects occurred locally in the tumors receiving i.l. injections and systemically, although to a slightly lesser extent, in the noninjected tumors of mice treated i.l. and in the xenografts of mice treated i.p. Biodistribution studies showed that the uptake of 125I-HuBrE-3vl/nIFN-alpha by the tumors 24 hours after i.l. or s.c. injection was greater than that of 125I-HuIgG1kappa/nIFN-alpha, 125I-nIFN-alpha alone, or by normal tissues, documenting a tumor targeting effect and favorable tumor:normal tissues (T:NT) ratios. The targeting effects and the resulting tumor growth inhibition were favored by the IFN-mediated up-regulation of the HuBrE-3vl reactive antigen, which was more prominent after 3 weeks of treatment with HuBrE-3vl/nIFN-alpha. These results were superior to those we obtained previously with nIFN-alpha conjugated to another MoAb of the same group (Mc5). These studies point out the potential usefulness of HuBrE-3vl/nIFN-alpha for the local and systemic treatment of breast cancer lesions by providing a means of delivering high doses of IFN to the tumors while minimizing the amount of IFN binding to normal tissues.

Regression of human breast cancer xenografts in response to intralesional treatment with interferons alpha and gamma potentiated by tumor necrosis factor.

The potentiating effects of human recombinant tumor necrosis factor-alpha (rTNF-alpha) on the antitumor actions of recombinant interferon-gamma (rIFN-gamma) and of natural interferons alpha and gamma combined (nIFN-alpha/nIFN-gamma) were studied on human breast cancer xenografts growing bilaterally in nude mice. The cytokines were injected singly or in combination in one of the two tumors of each mouse to study local effects while the opposite tumor was left undisturbed to evaluate systemic effects. The tumors received 20 intralesional injections (four cycles of 5 daily injections each). In injected tumors the best results were obtained with nIFN-alpha/nIFN-gamma supplemented with rTNF-alpha. The responses were dose dependent, resulting in complete regression of 9 of 9 tumors with rTNF-alpha used at the dose of 5 micrograms per injection, of 6 of 8 tumors at the dose of 2.5 micrograms, and of 4 of 8 tumors at the dose of 0.5 microgram. Mostly mild to moderate partial responses were seen in the other groups. The systemic effects on the contralateral tumors were significantly less than the local effects on the corresponding tumors. Histologically, responding tumors showed reactive fibrosis and inflammatory cell infiltration. No vascular alterations were seen, presumably because of the immunodeficiency of nude mice. It was concluded that the potentiation of the antitumor actions of IFNs by rTNF-alpha was effective at the local but not at the systemic level.

Regression of skin recurrences of breast carcinomas treated with intralesional injections of natural interferons alpha and gamma.

Two groups of patients with disseminated breast carcinomas who had failed radiotherapy, chemotherapy, and hormonotherapy were treated with natural interferon alpha (nIFN-alpha) alone or in combination with nIFN-gamma delivered in cycles of 10-12 intralesional (i.l.) injections to recurrent and metastatic lesions. In group, I, 16 skin lesions in 12 patients received nIFN-alpha alone resulting in 7 complete regressions verified histologically (CR), 7 partial regressions (PR), and no regressions (NR) in 2. Group II included 4 patients in whom 7 cutaneous recurrences were treated with nIFN-alpha/nIFN-gamma (5 CR, 2 PR), 2 were injected with nIFN-alpha alone (1 CR, 1 PR), and 1 received nIFN-gamma alone (PR). Two additional patients in group II were given i.l. injections of nIFN-alpha/nIFN-gamma to lymph node metastases (1 CR, 1 PR). Clinical toxicity was experienced by 5 of 12 patients in group I and by all the patients in group II and was controlled in most instances by antipyretics. Systemic antitumor effects were not appreciable clinically. Nevertheless, noninjected lesions exposed only to systemic levels of IFNs, when studied immunohistochemically, displayed an immunological response similar to that of IFN-injected lesions, although less intense. Therefore, IFNs can be useful in controlling locoregional recurrences of breast cancer even in patients who are not responding to other forms of therapy. Furthermore, in addition to the local antitumor actions, they appear to be capable of eliciting systemic immunological effects.

Injection of irradiated b16 melanoma genetically-modified to secrete IFN-alpha causes regression of an established tumor.

Highly aggressive murine B16 melanoma was engineered to secrete IFN-alpha constitutively. Cells expressing IFN-alpha were injected into syngeneic C57BL/6 mice and the mice were monitored for tumor development. Secretion of IFN-alpha by B16 melanoma cells completely abrogated their tumorigenicity in syngeneic mice. LFN-alpha-secreting cells also abrogated the tumorigenicity of IFN-gamma-secreting and TNF-alpha-secreting cells when injected in combination whereas cells secreting either IFN-gamma or TNF-alpha grow progressively in mice when injected alone. Moreover, protected animals developed significant immunity against subsequent challenge with parental cells. Injection of parental cells and IFN-alpha-secreting cells together in a mixed tumor transplantation assay resulted in a significant reduction of tumorigenicity of the parental cells. Histopathological studies of the tissues from the injection site of the mice inoculated with a combination of parental and B16.IFN-alpha cells revealed the existence of a massive cellular infiltrate composed of lymphocytes and granulocytes at an early stage (7-11 days). In the later stages (22 days), no recognizable tumor tissue was detected. Injection of irradiated IFN-alpha-secreting cells in the mice carrying an established tumor completely prevented tumor development in 80% of the treated mice when injection was performed on the same side as the tumors. Injection of irradiated IFN-alpha-secreting cells in the contralateral site showed much less effect on the established tumor. Systemic antitumor effects on the established tumor can be enhanced by using a combination of irradiated IFN-alpha and IFN-gamma secreting cells as a vaccinating inoculum.

IFN-gamma and b7 as costimulators of antitumor immune-responses.

We transfected the mouse IFN-gamma and/or the mouse B7 (T cell costimulatory molecule) cDNAs into B16 melanoma cells to study the effects of local constitutive expression of these molecules on the tumorigenicity and immunogenicity of this aggressive tumor. Cells expressing IFN-gamma (B16.IFN-gamma), B7 (B16.B7), B7 and IFN-gamma (B16.IFN-gamma/B7), and parental cells were injected subcutaneously (s.c.) into syngeneic C57BL/6 mice to compare their in vivo growth. We report that IFN-gamma secretion significantly reduced the tumorigenicity of B16 cells. These effects were related to the direct action of secreted IFN-gamma since i) in vivo injection of antiserum to IFN-gamma accelerated tumor growth, ii) development of tumor correlated with loss of IFN-gamma production, and iii) B16.IFN-gamma cells were tumorigenic in IFN-II receptor (IFN-gamma R) knockout mice, but not in parental mice. We propose that immune mechanisms are being activated by IFN-gamma since i) immune effector cells were recruited to the injection site, ii) expression of MHC class I and class II antigens was increased on cells secreting IFN-gamma and, iii) B16.IFN-gamma tumors appeared earlier in athymic mice than in immunocompetent mice. Since the in vivo growth of B16.IFN-gamma cells was not completely abolished, we studied the effect of co-expression of IFN-gamma and the T cell costimulatory molecule B7 on the tumorigenicity of B16 cells. We report that B16.IFN-gamma/B7 cells, which also express increased levels of MHC class I and class II molecules as compared to parental cells, had a dramatically suppressed tumorigenicity, while B16 cells expressing the B7 molecule only (B16.B7) were as tumorigenic as the parental cells. B16.IFN-gamma/B7 cells induced specific immune responses since all of the protected mice were able to reject challenges with parental cells. Results indicate that co-expression of two molecules which are involved in the activation of immune responses and in antigen presentation can influence the ability of the immune system to recognize and eliminate both transfected as well as parental tumor cell inocula and suggest that vaccines consisting of such cells may be used for the immunotherapy of cancer.

Up-regulation of a tumor-associated antigen (tag-72) by interferon-alpha and interferon-gamma in patients with cutaneous breast-cancer recurrences.

Up-regulation of a tumor-associated antigen (TAA) TAG-72 was investigated immunohistochemically in skin recurrences of 10 breast cancer patients treated with intralesional injections of natural interferons (nIFNs) alpha and gamma. Up-regulation was assessed by comparing the immunoreactivity of pre-IFN and post-IFN specimens stained with monoclonal antibodies (MoAbs) B72.3 and CC49 at near end-point dilutions. Varying degrees of enhanced immunoreactivity were detected in IFN-injected and non-IFN-injected lesions except in 1 patient with high constitutive expression of TAG-72 and another patient not expressing TAG-72. Thus, IFN-mediated up-regulation of TAG-72 can occur in patients whose breast cancer cells express this TAA, independently of the initial levels of antigenic expression.

Monoclonal antibody BrE-3 participation in a multivariate prognostic model for infiltrating ductal carcinoma of the breast.

Monoclonal antibody (MoAb) BrE-3, an anti-human milk fat globule (HMFG) MoAb, is used here as a novel prognostic indicator for survival and relapse time in patients with infiltrating ductal carcinoma of the breast. A scoring system (4-Score method) was developed to this effect that measured, in a statistically reliable fashion, the level of expression of the epitope for MoAb BrE-3 in the cytoplasm and membranes of breast carcinoma cells in paraffin-embedded sections. In univariate analysis, data obtained by the 4-Score Method as well as data from traditional prognostic indicators (tumor size, axillary node status, and grade of differentiation) were found to be associated with patient survival and relapse. In multivariate analysis, using a Cox proportional hazards regression model, levels of expression of BrE-3 epitope plus tumor size and axillary node status were weighted and combined in an Individual Linear Composite Prognostic Score (ILCPS) that had a high level of association with survival and relapse time in this sample model of patients with infiltrating ductal carcinoma of the breast. This level of association was found to be higher than the level of association for any other combination of traditional or 4-Score method variables. The level of expression of BrE-3 significantly adds to the prognostic capacity of traditional prognostic markers for infiltrating ductal carcinoma of the breast.

The use of natural interferon alpha conjugated to a monoclonal antibody anti mammary epithelial mucin (Mc5) for the treatment of human breast cancer xenografts.

An immunoconjugate composed of natural interferon alpha (nIFN alpha) bound in a noncleavable fashion to a monoclonal antibody (MoAb) recognizing a breast epithelial membrane mucin (Mc5) was used to to treat xenografts of a human mammary carcinoma cell line (MCF-7) growing in nude mice. The immunoconjugate (nIFN alpha/Mc5) was administered as 20 intralesional (i.l.) injections to 1 of 2 xenografts in each animal. It was found that nIFN alpha/Mc5 produced a significant enhancement of the growth inhibitory actions of nIFN alpha on the injected tumors. Further enhancement was obtained when nIFN gamma or nIFN gamma together with Mc5 (at a dose 10 times larger than that present in nIFN alpha/Mc5) were added to the immunoconjugate. Biodistribution experiments showed that the uptake of 125I-nIFN alpha/Mc5 by the tumors was greater and its elimination slower than for 125I-nIFN alpha alone or conjugated to irrelevant mouse IgG1. In addition, the immunoconjugate up-regulated the antigenic expression of a breast epithelial membrane mucin by the carcinoma cells, an up-regulation which was not significantly different from that produced by nIFN alpha alone. The contralateral noninjected tumors exposed to systemic levels of the immunoconjugate showed an enhancement of antitumor effects, but to a lesser extent than the injected tumors. These findings suggest that the enhancement of the growth inhibitory action of the immunoconjugate was related to the specific binding of Mc5 which targeted the IFN to the carcinoma cells and impeded its elimination. It is likely that the targeting was favored by the IFN-mediated up-regulation of antigenic expression by the carcinoma cells, thereby producing a cascade of interrelated effects. The results of this study point out the feasibility and potential usefulness of IFN treatment by means of immunoconjugates as well as the worth of pursuing and improving this form of therapy.

Cellular events accompanying regression of skin recurrences of breast carcinomas treated with intralesional injections of natural interferons alpha and gamma.

Cutaneous recurrences of breast carcinomas were treated with 10 i.l. injections of nIFNs alpha and gamma delivered in combination (7 lesions) or singly (11 with nIFN-alpha, one with nIFN-gamma). Histologically confirmed complete regressions occurred in 5 of 7 lesions treated with nIFN-alpha/nIFN-gamma and in 5 of 11 recurrences injected with nIFN-alpha alone. In all cases specimens were obtained before and after therapy. In addition, in some cases (4 treated with nIFN-alpha/nIFN-gamma, 2 with nIFN-alpha, one with nIFN-gamma) multiple recurrences were injected simultaneously and were excised 24 h after 1, 3, and 10 injections and 21 days after completion of therapy. The main findings observed in the treated lesions undergoing complete and partial regressions included: (a) inhibition of mitotic activity and up-regulation of antigenic expression (mammary epithelial membrane antigen, intercellular adhesion molecule 1, HLA-DR) by the carcinoma cells; (b) activation of macrophages and dendrocytes with marked expression of HLA-DR and HLA-A,B,C; (c) infiltration of the dermis and tumors by activated T-lymphocytes (CD3+, CD4+, CD8+); (d) questionable participation by B-lymphocytes and natural killer cells; (e) activation of endothelium with enhancement of antigenic expression (intercellular adhesion molecule 1, HLA-DR), procoagulant activity, and vascular permeability. The responses elicited by nIFN-alpha/nIFN-gamma were greater than those caused by either IFN used alone. It appears that in these patients the IFNs exerted an antiproliferative action and potentiated a cell-mediated immunological response liminally present in the neoplastic tissues prior to therapy.

Levels of expression of breast epithelial mucin detected by monoclonal antibody BrE-3 in breast-cancer prognosis.

Taking into consideration the relationship of breast neoplasia with recent knowledge obtained on the molecular structure and biosynthesis of the breast epithelial mucin, an epitope on this molecule detected by monoclonal antibody (MAb) BrE-3 was chosen as a marker to study the correlation of expression of the mucin and prognosis in infiltrating ductal carcinomas of the breast. Strong statistical validation was obtained in the use of BrE-3 in immunohistochemical procedures where scores for the expression of the mucin on paraffin-embedded sections of the primary breast tumors were studied. Four different immunohistochemical variables measuring levels of expression (intensity or prevalence) in cytoplasm or membrane were obtained for each of 227 patients' breast tumors and subjected to Kaplan-Meier univariate and Cox proportional-hazards multi-variate analysis. Additionally, traditional prognostic variables for breast-cancer prognosis (grade of differentiation, age, tumor size, axillary-lymph-node involvement and estrogen receptors) were subjected to identical analyses. In uni-variate analysis, low cytoplasmic intensity, high membrane prevalence, and high membrane intensity of mucin expression were each found to be significantly associated with good prognosis in relation to both survival or relapse time. In multi-variate analysis, all 4 immunohistochemical parameters were significantly associated with both survival and relapse time in these patients. Among the traditional variables, 3 (axillary-node involvement, grade of differentiation and tumor size) were also found to be statistically significant at the uni-variate and multi-variate level. A multi-variate analysis of the combined immunohistochemical and traditional variables identified the 4 immunohistochemical parameters, tumor size and axillary-node involvement as having the highest level of association with either survival or relapse time. These variables were then combined and served to define a prognostic model [ILCPS(Comb)], which was found to have the capacity to separate the patient population studied into 4 prognostic groups in terms of survival and 3 groups in terms of relapse. As expected, the ILCPS(Comb) was shown to have a higher level of prognostic association with both survival and relapse than the individual variables themselves, the traditional variables together or the immunohistochemical variables together. Our approach develops a theoretical framework and a statistical model, employing levels of expression of the breast epithelial mucin and 3 traditional variables, which identifies, in terms of prognosis, distinct sub-populations of patients with infiltrating breast carcinoma with defined risk functions.

An immunohistochemical evaluation of progesterone receptor in frozen sections, paraffin sections, and cytologic imprints of breast carcinomas.

Two monoclonal antibodies to progesterone receptor (PR), JZB39 and KD68, were used for the immunocytochemical visualization of PR in different kinds of breast cancer specimens including (1) cryostat sections of tumors frozen at -80 degrees C; (2) paraffin sections of tumors fixed in formalin or in Bouin's fixative for varying periods of time at room temperature or at 4 degrees C; and (3) imprints and cryostat sections prepared from the tissue used for frozen section diagnosis and stored at -80 degrees C after fixation in Zamboni's solution. Sections of conventionally frozen specimens as well as imprints and cryostat sections stored for varying periods of time were stained with the peroxidase-antiperoxidase technique, whereas the avidin-biotin technique was used for paraffin sections. In all types of specimens the PR immunostaining was localized to the nuclei of carcinoma cells and displayed considerable heterogeneity both in intensity and in distribution of positive cells. Close correspondence was found between the different immunohistochemical techniques as well as between immunostaining and steroid-binding assays. PR staining was more frequently positive in well-differentiated than in moderately or poorly differentiated carcinomas, whereas no meaningful correlation was found between PR staining and extent of the disease. Similar results were obtained with the immunostaining of estrogen receptor in the same material using monoclonal antibodies H222 and D75P3 gamma. Thus, by choosing the technique that best suits the type of specimen available, it is possible to obtain valid information on the receptor status of any breast carcinoma, regardless of its size and clinical presentation.

Antiproliferative effects of natural interferon beta alone and in combination with natural interferon gamma on human breast carcinomas in nude mice.

Nude mice bearing bilateral xenografts of human breast carcinoma cells (MCF-7 and BT20) were treated with 2 or 45-day cycles of intralesional (i.l.) injections of human natural interferon beta (nIFN-beta) alone or in combination with human natural interferon gamma (nIFN-gamma). The injections were administered to only 1 of the 2 tumors in each animal, thus making it possible to assess at the same time local therapeutic effects in the injected tumors and systemic effects in the contralateral ones. When n-IFN-beta was used as a single agent only mild local antitumor effects and virtually no systemic effects were observed. In contrast, the combined administration of nIFN-beta/nIFN-gamma produced marked antiproliferative effects, presumably as a result of the synergistic action of type I and type II IFNs. These effects ranged from complete regression documented histologically in 2 MCF-7 tumors to varying degrees of growth inhibition with persistence of residual microscopic or grossly detectable tumor. Local effects were more pronounced than systemic effects. The therapeutic efficacy of nIFN-beta proved to be greater than that of recombinant interferon beta (rIFN-beta). In MCF-7 tumors nIFN-beta appeared to be less effective than nIFN-alpha, whereas the opposite was true for BT20 tumors.

Malignant mesothelioma: diagnosis by skin biopsy.

We report a case of malignant mesothelioma in which the tissue diagnosis was established by examination of a skin biopsy specimen. We also review the cutaneous manifestations of malignant mesothelioma. To our knowledge this is only the second reported case diagnosed primarily by examination of tissue from the skin.

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.

Treatment of human breast cancer xenografts using natural interferons-alpha and -gamma injected singly or in combination.

Natural interferons (nIFNs) -alpha and -gamma were used to treat nude mice bearing bilateral xenografts of human breast cancer cells (MCF-7 and BT 20). The IFNs were administered singly or in combination by means of intralesional (i.l.) or intraperitoneal (i.p.) injections. In the animals treated intralesionally 1 of the 2 tumors was injected to study the local therapeutic effects, while the contralateral one was left undisturbed and used to assess systemic effects. Treatment of MCF-7 tumors with i.l. injections of nIFN-alpha and nIFN-gamma combined resulted in complete regression of the injected tumors in 8 of 20 mice treated for 2 weeks and in 10 of 10 mice of an additional group treated for 4 weeks. The corresponding contralateral tumors showed complete regression in 2 mice treated for 4 weeks and partial responses in the others. Incomplete responses were also observed when the IFNs were used singly or when they were delivered intraperitoneally. Similarly, in BT 20 xenografts the best results were obtained with i.l. injections of the 2 IFNs combined, but no complete regressions were achieved. These experiments provide further evidence for a synergistic interaction of nIFN-alpha and nIFN-gamma in vivo and indicate that the potentiated antitumoral activity is greater when these interferons are administered i.l.

Effects of intralesional injections of interferons-alpha on xenografts of human mammary carcinoma cells (BT20 and MCF-7).

The effects of intralesional injections of human natural and recombinant interferons-alpha (nIFN-alpha and rIFN-alpha A) were studied in nude mice bearing bilateral xenografts of human mammary carcinoma cells (BT 20, MCF-7). One tumor of each animal received intralesional injections, while the contralateral tumor was left undisturbed. Thus, the injected tumors were subjected to the local action of the IFNs whereas the opposite ones were exposed to the systemic effects of the IFNs seeping into the subcutaneous tissue following the intratumoral injection. When used singly these IFNs exerted an inhibitory effect on the growth of both injected and contralateral tumors, but failed to cause complete regression. Many of the cells of treated BT 20 xenografts showed significant morphological alterations (increased cell volume and nuclear pleomorphism) as compared to the untreated controls. Morphological alterations in MCF-7 tumors were difficult to assess because of the inherent pleomorphism of these cells. The immunoreactivity of BT 20 and MCF-7 tumors to monoclonal antibodies directed against milk fat globule proteins and against HLA antigens was not appreciably affected by treatment with these IFNs. This study confirms that intralesional injections of human IFNs-alpha partially inhibit the growth of human breast cancer xenografts, probably through a direct effect on the carcinoma cells. Under the present experimental conditions, the intralesional and the subcutaneous routes of administration appear to offer comparable antitumor effectiveness.

Immunostaining of estrogen receptor in paraffin sections of breast carcinomas using monoclonal antibody D75P3 gamma: effects of fixation.

Immunostaining of estrogen receptor was carried out on paraffin sections of breast carcinomas using an anti-estrophilin monoclonal antibody (D75P3 gamma) and the avidin-biotin technique. The tumors were fixed in Bouin's solution or in formalin for varying periods of time at room temperature or at 4 degrees C. Best results were obtained following fixation in Bouin's at room temperature or in formalin at 4 degrees C. The staining was localized in the nuclei of carcinoma cells and was heterogeneous in intensity and extent. Prolonged fixation resulted in decreased immunoreactivity and in the appearance of nonspecific cytoplasmic and background staining. The estrogen receptor immunostaining on paraffin sections was found to be in concordance with that on frozen sections (Abbott ER-ICA) and with the steroid-binding assay (dextran-coated charcoal) in over 90% of the cases. This method is of easy and rapid execution and yields reliable and reproducible results.

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.