Immunohistochemical heterogeneity of breast carcinomas negative for estrogen receptors, progesterone receptors and Her2/neu (basal-like breast carcinomas).

Basal breast carcinomas triple negative for estrogen receptors, progesterone receptors and Her2/neu breast carcinomas are more aggressive than conventional neoplasms. We studied 64 cases with immunohistochemistry, using 23 antibodies, to characterize diverse pathological pathways. A basal cytokeratin was identified in 81% of tumors and vimentin was identified in 55%. The mean Ki67 index was 46% (range, 10-90%). Coincident expression of p50 and p65, which suggests an active nuclear factor-kappaB factor, was present in 13% of neoplasms. Epithelial growth factor receptor (EGFR), insulin-like growth factor-I receptor (IGF-IR) or c-kit (CD117) was identified in 77% of tumors. Loss of protein tyrosine phosphatase was found in 14%, whereas Akt activation was present in 28%. Several differences were identified between two subtypes of basal breast carcinomas: the pure variant (negative S-100 and actin) was more frequently associated with 'in situ carcinoma' (P=0.019) and pBad overexpression (P=0.098), whereas the myoepithelial variant (positive S-100 or actin) showed more frequent tumor necrosis (P=0.048), vimentin expression (P=0.0001), CD117 expression (P=0.001) and activated caspase-3 (P=0.089). IGF-IR could be as important as EGFR for the growth of these neoplasms. Basal cell carcinoma has at least two subtypes with distinct microscopic and immunohistochemical features.     

E-cadherin quantititive immunocytochemical assays in breast carcinomas

The reduction of E-cadherin expression, which is involved in the initial step of invasion and metastasis of cancer, was investigated in 218 human breast carcinomas. Quantitative immunohistochemical assays (ICAs) were performed on frozen sections. Quantitation was assessed by processing digitized microscopic images of immunoreactions using a computerized system of image analysis (SAMBA). The results were correlated with clinicopathological data and quantitative immunodetection of other molecules. E-cadherin expression was significantly (P<0·001) stronger in ductal carcinomas than in lobular carcinomas and stronger (P<0·01) in low grades than in high grades, but E-cadherin was independent of lymph node status and tumour size. Also an inverse significant (P<0·01) relationship was observed between E-cadherin expression on tissue sections and positive immunoreactions with anti-P53, MIB1 (growth fraction), and anti-c-erb-B2 product. Conversely, strong positive and anti-E-cadherin immunoreactions correlated with strong positive anti-ER and anti-PR immunoreactions (P<0·01). No relationship was observed between E-cadherin and the results of quantitative ICAs of cathepsin D, CD31, and P-glycoprotein, assessed on consecutive sections from the same frozen tissue samples. The results show that preserved E-cadherin expression correlates with high degree of tumour differentiation, low proliferative activity, and low expression of prognostic markers. The deregulation of E-cadherin is independent of other steps of tumour invasion, such as protease digestion of extracellular matrix and angiogenesis. © 1997 John Wiley & Sons, Ltd.     

Immunohistochemical detection of estrogen and progesterone receptors in primary breast cancer

To evaluate the reliability of the immunohistochemical assay for estrogen receptor (ER) and progesterone receptor (PR) in the prognosis of patients with breast cancer, 83 primary tumors from the patients were studied. Immunohistochemical analysis was performed using antibody ER 1D5 for ER determination and antibody PR-ICA for PR determination. Of all tumors, ER and PR positivities were detected in 36.1% and 45.8% respectively. There was no significant relationship between ER, PR and age of the patients, tumor size or number of involved nodes. However, we found that only the immunohistochemical ER was a predictor of early recurrence in patients with primary breast cancer. In addition, there was no additive effect in recurrence-free survival when both receptor expressions were combined.     

Quantitation of estrogen and progesterone receptors by immunocytochemical and image analyses.

Compared to other techniques, the ability to detect estrogen and progesterone receptors by immunocytochemical analysis in formalin-fixed, paraffin-embedded sections has clear advantages, including the ability to assay small biopsy specimens, fine-needle aspirate samples, and archival material. Twenty-two cases of breast carcinoma were evaluated for estrogen and progesterone receptors by immunocytochemical analysis and enzyme immunoassay. Using a true color-based image analysis system, histograms of area versus the optical density of the positive staining nuclei were generated. A binary decision algorithm was derived from these histogram parameters by the CART computer program. Estimates generated by the algorithm for image analysis/immunocytochemical analysis had a 90% concordance with the enzyme immunoassay values. It is concluded that quantitative immunocytochemical results for estrogen and progesterone receptor content in formalin-fixed, paraffin-embedded tissue can be generated using image analysis.     

Immunocytochemical detection of estrogen and progesterone receptors in 124 human breast cancers

Monoclonal antibodies to human estrogen receptor (ER), and rabbit progesterone receptor (PR), also recognizing human PR, were used to detect the receptors by peroxidase immunocytochemistry in frozen sections of 124 primary breast carcinomas. Both ER and PR were almost exclusively located in carcinoma cell nuclei, with heterogeneous distribution and intensity. The staining results were evaluated semiquantitatively (histoscore), based on the percentage of positively stained carcinoma cells and nuclear staining intensity. The receptor status thus determined was as follows: ER+PR+ in 50 patients, ER+PR- in 23, ER-PR- in 26, and ER-PR+ in 3 patients. There was a 79% (ER) or 70% (PR) agreement in the positivity/negativity between the immunocytochemical and steroid-binding assay (in 102 patients) with a highly significant correlation. The histoscore values increased significantly with cytosol receptor levels (ER, r = 0.623, P less than 0.001; PR, r = 0.366, P less than 0.01).     

Fluorescent cytochemical detection of estrogen and progesterone receptors in breast fine-needle aspirates

Estrogen and progesterone receptors were studied in fine-needle aspiration biopsy specimens of 56 patients with primary, recurrent, or metastatic breast carcinoma. The ligands, 17 B-estradiol-6-carboxymethyloxine-bovine serum albumin fluorescein isothiocyanate (FITC-BSA estradiol) and hydroxyprogesterone hemisuccinate bovine serum albumin tetramethyl rhodamine isothiocyanate (TMRITC-BSA progesterone), were used in the fluorescent cytochemical method. The findings obtained from the aspirated cells with the use of the fluorescent cytochemical technique were compared with results obtained from the cell population of the same tumor after removal with the use of both the fluorescent cytochemical technique and the biochemical dextran-coated charcoal (DCC) assay. For the needle aspirates, there was 89% concordance for estrogen receptor and 86% concordance for progesterone receptor between biochemical and cytochemical results. A high degree of correlation was also demonstrated between fine-needle aspirates and imprint preparations with the use of the cytochemical technique. This study suggests that the fluorescent cytochemical technique is an effective tool in assessment of estrogen and progesterone receptor content in fine-needle aspirates of primary and metastatic breast cancer. The fluorescent cytochemical technique can be performed easily at community hospitals and is well suited for specimens of insufficient size for biochemical assay.     

Immunocytochemical analysis of receptors for estrogen and progesterone in fine-needle aspirates from human breast carcinomas

Monoclonal antibodies were used for a preoperative analysis of estrogen receptors (ERs) and progesterone receptors (PgRs) in fine-needle aspirates from 44 primary human breast carcinomas. The semiquantitative receptor values obtained in cytologic specimens correlated well with those from enzyme immunoassay analysis on surgically removed tumor tissue (r = 0.74 for PgR; r = 0.75 for ER). Cytologic smears showed a heterogenous tumor cell distribution of ER and PgR in 29 and 23 cases, respectively. The results suggest that measurement of the ER and PgR in cytologic smears is an accurate and reliable technique that can be performed on a minimum amount of tissue.     

Potential value of estrogen receptor immunocytochemical assay in formalin-fixed breast tumors.

Sixty-two primary breast carcinomas were analyzed for estrogen receptor (ER) by both the dextran-coated charcoal (DCC) technique and estrogen receptor immunocytochemical assay (ER-ICA) on cryostat and permanent sections. Paraffin sections of formalin-fixed breast tissue underwent DNase pretreatment to expose the nuclear antigenic site as described by P. Shintaku and J. H. Said (Am J Clin Pathol 87:161, 1987). The results of immunocytochemical staining agreed with those of the DCC biochemical assay in 89% of paraffin-sectioned tissue and in 94% of the cryostat sections. Comparison of the results of ER-ICA on permanent and frozen sections showed 85% agreement (kappa statistic = 0.704). This study suggests that ER can be demonstrated immunocytochemically on paraffin-sectioned breast tissue. However, although highly specific, immunoperoxidase determination on paraffin-embedded tissue is less sensitive than that on frozen tissue. The commercial source of DNase, length of incubation, and tissue fixation are important factors in the demonstration of ER immunoreactivity. The assay may offer an alternative for assessment of ER when tissue is not suitable or available for biochemical assay or conventional cytochemical analysis.     

Automated immunohistochemical estrogen receptor in fixed embedded breast carcinomas.

The authors immunohistochemically assessed the presence of estrogen receptor (ER) in formalin-fixed, paraffin-embedded tissue sections of 68 breast carcinomas by an automated method using Pronase (CalBiochem, La Jolla, CA) predigestion and alkaline phosphatase detection (Method 1). These results were compared with those obtained by an automated peroxidase-antiperoxidase method with DNAse pretreatment of fixed embedded sections (Method 2), with ER immunostain on frozen sections (Method 3), and with biochemical results (dextran-coated charcoal cytosolic [DCC] assay). Compared with the DCC assay, Methods 1, 2, and 3 gave sensitivities of 54%, 25%, and 89%, respectively. The sensitivity for Method 1 was increased to 74% in those cases with DCC results showing greater than 50 fmol/mg protein. These findings indicate that ER immunohistochemical studies on formalin-fixed paraffin-embedded tissues (as assayed by Method 1) provide useful clinical information when the results are positive. A negative result, especially if surrounding normal elements are not positive, may indicate no receptors, receptor levels less than 50 fmol/mg protein, or improper tissue preservation. In the absence of fresh tissue for ER assay by DCC assay or of frozen sections for immunostaining, and with an understanding of its limitations, this method may be useful.     

Pulmonary lymphangioleiomyomatosis and steroid receptors. An immunocytochemical study

Estrogen and progesterone receptors were demonstrated immunocytochemically in the lung tissue of two cases of pulmonary lymphangioleiomyomatosis. In one case both receptors were localized in the proliferative smooth muscle nodules and diffusely in the interstitial tissue in close proximity to the muscle. The pulmonary tissue in the other case contained the estrogen receptor only. The authors conclude that the response to hormonal treatment seen in this disease might be optimized in the future by monitoring for the presence of steroid hormone receptors.     

A comparative immunohistochemical and biochemical study of estrogen receptors in breast cancer

The authors review the results of a comparative study of 25 mammary carcinomas, examined by the immunohistochemical method (IHM) with the use of ER-D5 monoclonal antibodies (Amersham) and by the cytosol receptor protein binding of a labeled ligand followed by the unbound hormone adsorption on dexteran-coated coal (DCM). Pap test with the above antibodies has been employed in immunohistochemical studies. The IHM has been highly specific. The results coincided in 21 of the 25 cases (83.3%). The reaction has been heterogenous in the same tumor. Quantitative values obtained by the two methods coincided in many cases. The IHM may have a wide practical application, provided the reagents are available, for it does not require sophisticated equipment and is simple to perform.     

Immunohistological study of oestrogen receptors in breast carcinomas that are biochemically receptor negative.

The reliability of an immunohistological method, applied to paraffin wax sections, was assessed for determination of oestrogen receptor content of biochemically oestrogen receptor negative breast carcinomata. Sixty consecutive tumours with oestrogen receptor concentrations of less than 10 fmol/mg cytosol protein, as estimated by dextran-coated charcoal biochemical assay, were examined. Paraffin wax sections were treated with DNAse before applying a peroxidase-anti-peroxidase method using ER-ICA monoclonal antibodies. Fifty one cases (85%) were negative, six (10%) weakly positive, and three (5%) were moderately positive. No strongly positive cases were seen. It is suggested that cases with weakly positive staining, especially when localised to a small area, should be regarded as negative. On the other hand, as the three moderately stained cases included two small tubular carcinomas and an invasive ductal carcinoma with high progesterone receptor concentrations, it is more likely that the biochemical assay in these cases represented false negative results due to sampling error or inclusion of fibrous or other non-neoplastic tissue in the assayed samples. It is concluded that the immunohistological method used here is fairly reliable and would be especially valuable for determination of oestrogen receptor content in small, mammographically detected tumours from which no tissue would be available for biochemical assay or frozen section examination.     

Immunohistochemical detection of estrogen receptors in paraffin sections from primary and metastatic breast cancer

With the availability of monoclonal antibodies against the estrogen receptor (ER) it is possible to demonstrate the presence of ER immunohistochemically. Some of the antibodies are claimed to be reactive in formalin fixed, paraffin embedded tissue. We have evaluated the reactivity of one of these antibodies, D75 and found an acceptable reaction in routinely formalin fixed, paraffin embedded tissue. The antibody was applied to both primary and secondary tumors from a group of patients with recurrent breast cancer. The metastatic lesions consisted of lymph node metastases, bone marrow metastases, and liver metastases. While 41% of the primary tumors were ER-positive, this was only the case with 35%, 20%, and 17% of the lymph node, bone marrow, and liver metastases, respectively. The discordance between the ER-status of the primary tumor and the distant metastasis was 41% in cases of bone marrow metastases, and 44% in liver metastases. In most cases the shift was from an ER-positive primary tumor to an ER-negative metastasis. The results support the hypothesis that ER-negative tumor cells are probably more aggressive with a larger metastatic potential than the higher differentiated, ER-positive tumor cells.     

Imprint cytology in immunocytochemical analysis of oestrogen and progesterone receptors of breast carcinoma.

Cytological imprint material from 26 mammary carcinomas was stained with monoclonal antibodies to oestrogen and progesterone receptors in an immunoperoxidase procedure. The staining result was compared with that of parallel stainings of frozen tissue sections of the same tumours. The peroxidase reactions in both techniques were semiquantitatively assessed (histoscore). In both sets of stainings the results agreed in 25 of 26 cases (oestrogen receptor: 19 positive, six negative; progesterone receptor: 14 positive, 11 negative). The histoscores of imprint preparations and cryostat sections showed a significant correlation in linear regression analysis (oestrogen receptor: r = 0.755, p less than 0.001; progesterone receptor: r = 0.740, p less than 0.001). Imprint cytology is simple, does not require expensive instruments, and no separate specimen has to be sequestered. It is especially suitable for immunocytochemical steroid receptor analysis of small breast carcinomas.     

Correlation of HER-2 status with estrogen and progesterone receptors and histologic features in 3,655 invasive breast carcinomas

We studied the inverse relationship between HER-2 and estrogen (ER) and progesterone (PR) receptors using HER-2 testing and correlated HER-2 status with histologic features in 3,655 unselected invasive breast carcinomas. Immunohistochemical analysis for ER, PR, and HER-2 and fluorescence in situ hybridization for HER-2 were performed. ER and PR expression were decreased significantly in HER-2+ tumors compared with HER-2- tumors (ER, 49.1% vs 78.17%; PR, 24.3% vs 53.13%). Even among HER-2+ tumors, the rate of ER or PR expression in high-grade tumors was significantly decreased compared with intermediate-grade tumors. HER-2 was positive in 10.87% of grade 2 and 27.84% of grade 3 ductal carcinomas and negative in all grade 1 ductal carcinomas. HER-2 overexpression or amplification essentially was limited to grades 2 and 3 ductal carcinomas and correlated inversely with ER or PR expression. Although ER or PR expression is decreased in HER-2+ tumors, a substantial proportion of them still express ER or PR.     

Immunohistologic detection of estrogen receptors in paraffin-embedded breast cancers: correlation with cytosol measurements

Paraffin-embedded sections of 76 human breast tissue specimens were analyzed for estrogen receptors (ER) and endogenous bound estrogen (ER-E). Preincubation of sections with polyestradiol phosphate was followed by stabilization of the complex with glutaraldehyde. The bound hormone was then visualized by the peroxidase-antiperoxidase (PAP) technique with antiestradiol as the primary antiserum. Normal breast tissue and benign proliferations were consistently positive for ER and ER-E. All specimens were examined for free and bound receptors in cytoplasm and nuclei. Among the carcinomas examined, a high correlation was found between the presence of ER by the PAP method and by the biochemical analysis of cytosol preparations. The PAP method, requiring no special preparation of surgical specimens, overcomes many of the disadvantages of the cytosol method and adds the advantage of independent evaluation of nuclear and cytoplasmic estrogen binding sites.     

Comparison of immunoexpression of 2 antibodies for estrogen receptors (1D5 and 6F11) in breast carcinomas using different antigen retrieval and detection methods.

The importance of in situ immunodetection of hormone receptors for therapy planning and prognostic evaluation in patients with breast carcinoma is well established. Sensitive detection methods are of utmost importance, especially in poorly fixed tissues, which are not uncommon in routine pathologic practice. The purpose of the present study is to compare immunoexpression of estrogen receptors in 20 cases of invasive ductal carcinoma using two antibodies, 1D5 and 6F11, and to verify the effect of different antigen retrieval solutions and detection systems. Immunoperoxidase was performed on paraffin sections using 1D5 and 6F11 as primary antibodies. Heat-induced antigen retrieval was performed using citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 8.9). Detection was achieved using the following systems: EnVision, EnVision Plus, and labeled streptavidin-biotin peroxidase complex. Reaction was semiquantified from 0 to 4. There were no differences between the two markers, 1D5 and 6F11, except when 6F11 was used with EnVision and citrate buffer, in which case weaker reactivity was observed. Only in this combination (6F11/EnVision) was EDTA buffer significantly better than citrate. Labeled streptavidin-biotin peroxidase complex presented the best results, followed by EnVision Plus.     

G-cell hyperplasia in chronic hypercalcemia. An immunocytochemical and morphometric analysis

The distribution of antropyloric G cells was mapped immunocytochemically and quantitated morphometrically in chronically hypercalcemic and normocalcemic patients. In the normocalcemic (control) patients, the G-cells were sparse in number and confined to the lower third of the antropyloric mucosa, where they were distributed singly or in small clusters within the glands. The absolute G cell (AGC) counts were 5.8 +/- 0.26 (mean +/- SE) per 0.25 sq mm of mucosa. The hypercalcemic patients showed a marked increase in their antropyloric G-cell population. The cells were uniformly distributed throughout the lower two-thirds of the mucosal thickness and were present in large numbers in practically every gland. The AGC counts in these hypercalcemic patients were 48.2 +/- 13.0, a statistically significant increase. These observations indicate that in man chronic hypercalcemia of diverse etiology is associated with antropyloric G-cell hyperplasia. The physiologic significance of this finding and its role in the pathogenesis of peptic ulcer disease needs to be elucidated.     

Lectin binding and steroid receptors in human breast carcinomas

A series of breast carcinomas of known steroid receptor status have been examined for evidence of binding of the lectins peanut agglutinin, soy bean agglutinin and wheat germ agglutinin. Correlations were found between oestrogen receptor status and reactivity of carcinomas to peanut agglutinin and soy bean agglutinin but these were not absolute. Wheat germ agglutinin binding was unrelated to the presence of oestrogen receptors. No relationship was evident between progestogen receptors and the binding of any lectin. It therefore seems unlikely that lectin histochemistry can replace steroid receptors as markers of hormone dependence in breast carcinomas.