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.     

Assessment of oestrogen receptor content of breast carcinoma by immunohistochemical techniques on fixed and frozen tissue and by biochemical ligand binding assay.

The oestrogen receptor content of 61 breast carcinomas was assessed by biochemical ligand binding assay and three immunohistochemical techniques--a frozen section method (Abbott ER-ICA) and on paraffin wax sections after fixation by two methods. The two fixatives used were Carson's buffered formalin and methacarn, and a DNAse pretreatment of sections was used. Overall agreement for the immunohistochemical methods with the ligand binding technique were 95%, 85%, and 86% for the frozen, formalin, and methacarn methods, respectively. A semiquantitative staining score was performed and all three methods gave significant correlations of staining scores with biochemical ligand binding values. The frozen section method was best (r = 0.88) with the fixed tissue methods yielding poorer correlation coefficients. Several factors affected staining, including the nature of the fixative and variable activity of DNAse. It is concluded that immunohistochemical assessment of oestrogen receptor content on fixed tissue provides acceptable qualitative information but that standardisation of protocols for tissue processing will be necessary for optimal utility and especially for quantitative assessments.     

Oestrogen receptor staining of paraffin-embedded breast carcinomas following short fixation in formalin: a comparison with cytosolic and frozen section receptor analyses

This paper describes an improved immunohistochemical method for demonstrating oestrogen receptor (OR) protein in paraffin-embedded sections of tissue fixed for 1.5 h in formalin. Thirty-two cases of infiltrating ductal breast carcinoma were stained with a monoclonal anti-OR antibody (H222), using a standard streptavidin-biotin method, following pretreatment with pronase. OR counts in paraffin sections were compared with those of frozen sections and with cytosolic values determined by a dextran-coated charcoal method. Twenty-seven of the carcinomas were OR-positive in paraffin sections. There was concordance between the paraffin section and the frozen section-determined receptor status in 30 cases (94 per cent) and a strong correlation was observed (r = 0.76; P less than 0.0001). Similarly, OR counts in paraffin sections correlated with cytosolic OR values (r = 0.60; P less than 0.001) and there was concordance in 97 per cent of cases. The percentage of positively-stained tumour cells in paraffin sections ranged from 0 to 94 per cent with staining intensities comparable to those seen in frozen sections. Staining of paraffin sections identified more OR-positive tumours than either frozen section staining or cytosolic assay. This study validates immunohistochemical OR analysis in formalin-fixed, paraffin-embedded breast carcinomas using a commercial anti-OR antibody.     

Detection of estrogen receptors with monoclonal antibodies in routinely processed formalin-fixed paraffin sections of breast carcinoma. Use of DNase pretreatment to enhance sensitivity of the reaction

This study describes an improved immunohistochemical method for the sensitive and specific identification of estrogen receptors (ERs) in paraffin sections from formalin-fixed and routinely processed breast carcinoma tissues, using DNase pretreatment to expose nuclear antigenic sites and commercially available immunoreagents (including monoclonal antibody) in kit form. Results were compared with dextran-coated charcoal cytosolic assay (DCC) and with conventional immunohistochemistry on frozen sections. Sensitivity and specificity for determinations on paraffin sections were 88% and 86%, respectively, and statistical analysis showed very good agreement between DCC and paraffin sections (kappa = 0.805). The DNase technic on paraffin sections allows excellent correlation between histologic characteristics and ER status and reduces DCC sampling error resulting from stromal dilution and tumor variability. This method offers a reliable and reproducible alternative when tissue is not suitable or unavailable for DCC or frozen tissue analysis and can be used for retrospective studies on stored tissue blocks.     

The value of tumour marker CA 125 in surgical pathology

CA 125 is a tumour marker located primarily on non-mucinous epithelial ovarian tumours and which is recognized by the monoclonal antibody OC 125. In this study the value of CA 125 in surgical pathology was assessed. In fresh frozen material, the expression of CA 125 was demonstrated in 82% of 83 epithelial ovarian neoplasms using the indirect immunoperoxidase technique. In addition, all adenocarcinomas of cervix (n = 5) and endometrium (n = 15) tested expressed CA 125, and 25 of 111 (22%) non-gynaecological malignant tumours were positive. The positive cases included 20 breast carcinomas, one carcinoma of the stomach and one of the colon. Using a commercial kit on routinely fixed, paraffin embedded material, CA 125 positivity was demonstrated in 29 of 36 (80%) serous cystadenocarcinomas after pronase pre-treatment of the sections, in contrast to 100% (n = 25) positivity on frozen tissue sections. CA 125 can, therefore, be demonstrated in routinely fixed paraffin embedded material, although the number of positive results is less than in fresh frozen sections.     

Surface membrane staining of immunoglobulins in paraffin sections of non-Hodgkin's lymphomas using immunogold-silver staining technique.

The immunogold-silver staining (IGSS) method is a new immunostaining technique with much enhanced sensitivity for demonstration of antigens in paraffin sections. A series of 10 non-Hodgkin's lymphomas of B cell type were stained for surface membrane immunoglobulins by the IGSS and peroxidase-antiperoxidase (PAP) methods using paraffin sections and polyclonal primary antisera. The resulting staining patterns were compared with those obtained using frozen sections of the same tissues, monoclonal antibodies and the immunoperoxidase technique. The IGSS method gave a clear demonstration of surface membrane immunoglobulins in neoplastic lymphocytes using paraffin sections and the pattern of staining achieved was comparable to that obtained by the immunoperoxidase technique employing frozen sections and monoclonal antibodies. PAP staining of paraffin sections consistently failed to demonstrate the presence of any surface membrane immunoglobulin. The IGSS method provides a new approach to the diagnosis of B cell lymphomas in which routinely fixed and processed tissues may be employed to demonstrate monoclonality.     

Immunohistochemistry of estrogen receptor protein in paraffin sections. Effects of enzymatic pretreatment and cobalt chloride intensification

Monoclonal antibodies provide important tools for the demonstration of estrogen receptors (ERs) in cases of breast cancer. This study reports an improved immunohistochemical method for the demonstration of ER in formalin-fixed paraffin-embedded tissue samples using the Abbott monoclonal antibody to ER protein. Tissue sections were pretreated briefly with trypsin, followed by DNase before the performance of the immunohistochemical reaction and cobalt chloride was used to intensify the color of the diaminobenzidine reaction product. In 20 cases, the results in paraffin sections were compared with biochemical assays with the dextran-coated charcoal technique or with immunohistochemistry performed on frozen sections. There was an excellent correlation between the results obtained with all three methods. The introduction of cobalt chloride into the chromogen solution significantly increased the sensitivity of this approach as compared with the use of diaminobenzidine alone.     

Immunocytochemical analysis of progesterone receptors in breast cancer.

Breast cancer specimens from 116 patients were assayed for the presence of progesterone receptor (PR), with the use of a highly specific monoclonal antibody and the peroxidase-antiperoxidase technique on cryostat and permanent sections. Results were compared with those obtained by the conventional PR determination by dextran-coated charcoal (DCC) assay; they were in concordance in 90% of cryostat sections and 85% of paraffin-embedded tissue. The sensitivity and specificity of the PR immunocytochemical assay (PR-ICA) were 91% and 89% for frozen sections and 83% and 89% for permanent sections, respectively. The immunostained slides also were evaluated for several semiquantitative features, including staining intensity, heterogeneity of staining, and the proportion of positive tumor cells. A statistically significant correlation was found between the percentage of tumor cells stained with the PR immunocytochemical technique and the PR-cytosol levels (P less than 0.05). These results suggest that the PR-ICA is an effective tool in the evaluation of PR content in breast cancer and can be applied in paraffin as well as frozen sections. This technique provides excellent morphologic detail, as well as tissue localization for PR. It also offers an alternative for assessment of PR when fresh tissue is not available for conventional hormone receptor analysis. The immunocytochemical assay can be performed easily at community hospitals. Because it requires only a small amount of tissue, PR-ICA is an ideal method for analyzing specimens of insufficient size for the DCC assay. This technique also is suited to the evaluation of fine-needle aspiration biopsy specimens.     

Semiquantitative analysis of the effects of fixation and paraffin embedding on immunoreactivity of renal basement membranes to a monoclonal antibody against type IV collagen.

The effects of formalin fixation and paraffin embedding on the immunoreactivity of human kidney to a monoclonal anti-type IV collagen antibody (JK-199) were examined semiquantitatively by a modified enzyme-linked immunosorbent assay (ELISA). The intensity of immunoreactivity in paraffin sections of the tissue fixed overnight with 10% formalin was approximately 70% of that in frozen sections. Immunoreactivity reduced to this extent did not impair the specific staining of basement membranes. Paraffin sections of tissues fixed 2 days showed 50-60% of the reactivity in the frozen sections of the tissue fixed overnight; the basement membranes in Bowman's capsules were stained positively, but those in other sites were not. The paraffin sections of tissues fixed 7 or 14 days showed no specific immunostaining. The immunoreactivity for type IV collagen in the basement membranes was restored after treatment with pronase E. The immunoreactivity after the enzymatic treatment was about 150% of that in the frozen sections of the overnight fixed specimens.     

Immunocytochemical staining of progesterone receptor in paraffin sections of human breast cancers.

Monoclonal antibodies (mAB) against progesterone receptor (PR) and the peroxidase-antiperoxidase (PAP) method to visualize PR in paraffin sections from 68 human breast cancers were used. Ten mAB, which recognize human PR on frozen sections, were tested. Six could detect PR in paraffin sections, with Li 417, LET 456, and LET 126 giving the best results. LET 126 antibody was used for most further studies. The effects of fixation with picric acid-formaldehyde (PAF), buffered-formalin, or with Bouin solution were investigated; all fixation methods allowed PR immunolabeling, although PAF or buffered formalin usually gave the best results. Positive staining was seen in the nucleus of carcinoma cells. Variations in intensity and extent of immunoreactivity were observed in all sections and among different regions of the same specimen. These were probably related to the heterogeneity of the tumor cell population. Results were compared with the PR content in the respective tumor tissues, determined by steroid-binding assay, and with immunocytochemistry on frozen sections. It was shown that there were correlations between the immunocytochemical staining (positive or negative) and the steroid binding assay (80%) and between the immunocytochemical staining on paraffin sections and on frozen sections (78%). Weaker intensity and fewer number of PR-positive cells were found for paraffin-embedded tumors. Estrogen receptors were also detected on adjacent sections from the same paraffin-embedded tissues by use of monoclonal anti-ER antibodies (ERICA-kit[Abbott Laboratories, Chicago, IL]) and DNase pretreatment. In conclusion, this immunocytochemical method for detection of PR and ER on paraffin sections offers an alternative to frozen tissue. It allows histologic and immunocytochemical studies on the same sample and retrospective studies on stored tissue blocks.     

Demonstration of estrogen receptor by immunohistochemical staining in paraffin sections of breast carcinoma

Paraffin embedded sections of 64 breast carcinomas were stained immunohistochemically using a commercially available monoclonal antibody to estrogen receptor. To improve the sensitivity of the staining, the authors used a Pronase enzyme pretreatment, biotinylated antibody to rat IgG as secondary antibody, streptavidin-alkaline phosphatase as tertiary reagent and fast red as chromogen. When compared to the results of estrogen receptor enzyme immunoassay, this method yielded an 85.9% concordance rate, 86.2% specificity and 85.7% sensitivity. When compared to estrogen receptor immunocytochemistry(ER-ICA) in frozen section and considering the inherent advantages of immunohistochemical staining over biochemical assay, the major advantages of this method are good morphology, suitability for retrospective study and reduced cost of staining due to dilution of expensive primary antibody. Thus, this method offers an alternative to ER assay using fresh tissue and should provide additional valuable information about estrogen receptor.     

Immunohistochemical estrogen receptor assay: quantitation by image analysis

The immunohistochemical estrogen receptor (ER) assay quantitated by image analysis was compared with the biochemical dextran-coated charcoal (DCC) assay in 97 primary breast carcinomas. Frozen sections immunostained for ER with a monoclonal antibody (ER-ICA; Abbott Laboratories, Chicago, IL) and the peroxidase-antiperoxidase technique were scored semiquantitatively by microscopy (HSCORE) and quantitatively [percentage of nuclear area immunopositivity (PNA)] using the CAS 200 image analyzer (Elmhurst, IL). There was a significant and identical correlation between DCC and both HSCORE and PNA. According to sensitivity/specificity calculations, the cut-off points of 10 for HSCORE and 1% for PNA were chosen with positive and negative predictive values of 97.7% and 74.5%, respectively. Low DCC-positive cases between 10 and 50 fmol of ER/mg of protein were immunohistochemically ER positive only in 61.5%. Immunohistochemical results of greater than 150 HSCORE and greater than 15% PNA corresponded to high DCC results of greater than 150 fmol/mg of protein. Immunohistochemical assay quantitated by image analyzer (PNA) is a comparable alternative to biochemical ER, especially when insufficient fresh tissue is available.     

Detection of surface antigen 17-1A in breast and colorectal cancer

Substantial progress has been made in detecting cell surface or intracytoplasmatic antigens to identify spread tumor cells with monoclonal antibodies (MAbs). The 17-1A antigen is already used as a target for specific immunotherapy in colorectal cancer. The purpose of this study was to compare the expression of 17-1A antigen in colorectal tumors versus breast cancers. MAb against the epithelial-specific antigen (ESA) and a routine staining technique were used to detect the 17-1A antigen in 100 cases of colorectal and 111 cases of breast cancer. The antigen expression of each tumor entity was examined by light microscopy on paraffin sections. Thirty six of the formalin-fixed paraffin sections of breast cancer were compared with their corresponding frozen sections. Evaluation was realized by a histological score (grade 0-9) considering the distribution and the staining intensity. We found an antigen expression of 17-1A in colorectal cancer quantified at 7.1+/-1.8 and at 4.5+/-2.5 for breast cancer in our score. Comparing paraffin sections and frozen sections in the 36 cases of breast cancer, the score was 5.5+/-2.3 in the paraffin and 8.1+/-1.9 in the frozen section group. Our results confirmed the high expression of 17-1A cases of in colorectal carcinoma. Furthermore, 17-1A is expressed in the majority of breast carcinomas, revealing a high difference between paraffin and frozen sections. As a result, a specific immunotherapy with MAbs against 17-1A antigen in minimal residual stages of breast cancer might be considered.     

Oestrogen and progesterone receptors in screen-detected breast carcinoma: an immunohistological study using paraffin sections

The presence of oestrogen and progesterone receptors was studied in paraffin sections of 81 screen-detected breast carcinomas using the monoclonal antibodies ER-ICA and PgR-ICA (Abbott) and the immunoperoxidase technique. The immunohistological results were compared with the results of the standard dextran-coated charcoal biochemical assay in 28 tumours which were big enough to provide tumour tissue for this assay. Sixty-three cases (78%) were oestrogen receptor positive and 62 (77%) were progesterone receptor positive. There was no statistical difference between receptor positivity in palpable or impalpable, in situ or invasive tumours. In the 28 cases where the biochemical assay was carried out, the two methods gave similar results in 23 (82%) and 21 (75%) tumours for oestrogen and progesterone receptors respectively. The majority of the remaining tumours, with one exception, were positive with immunohistology and negative with biochemistry. A good correlation was also present between the mean numerical biochemical values and the semiquantitative histological scores for both receptors. It is concluded that assessment of receptor status of small screen-detected carcinomas is feasible using routinely processed paraffin sections. There is reasonably good correlation with the results obtained by the standard dextran-coated charcoal biochemical assay, but more genuine receptor positive cases are detected by immunohistology.     

Demonstration of oestrogen receptors in paraffin wax sections of breast carcinoma using the monoclonal antibody 1D5 and microwave oven processing.

This study aimed at assessing the usefulness of a new monoclonal antibody (1D5) for the demonstration of oestrogen receptors (ER) in paraffin wax sections, using brief microwave processing rather than proteolytic predigestion. Routinely processed paraffin wax sections of 50 cases of breast carcinoma with known ER concentrations, estimated by the standard dextran-coated charcoal (DCC) biochemical assay, were examined using the avidin-biotin complex-immunoperoxidase technique. The results were assessed semiquantitatively, using a five grade scoring system. Of the 50 cases examined, 37 were positive and six were negative by both DCC and immunohistology. Of the remaining seven cases, three (6%) were negative by DCC but positive with immunohistology, and four (8%) were positive with DCC and negative with immunohistology. The DCC results of the latter four cases were 10, 14, 14 and 16 fmol/mg protein which is at the lowest level of positivity, our cutoff point being less than 10 fmol. The monoclonal antibody 1D5, as used in this study, can provide easily assessed reliable information about the ER status of breast carcinoma using routinely processed paraffin wax sections.     

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%.     

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.     

Vimentin expression in benign and malignant breast epithelium.

AIMS--To determine vimentin expression in epithelial cells in benign breast disease and malignant breast tumours; to assess the value of vimentin expression as a prognostic indicator in breast carcinoma. METHODS--Frozen and formalin fixed, paraffin wax embedded sections from 78 carcinomas, three phyllodes tumours, 19 fibroadenomas and 19 cases of fibrocystic disease were examined with a monoclonal antibody from the V9 clone. A correlation between vimentin expression and known prognostic indicators was sought in ductal carcinomas. The intracellular localisation of vimentin was examined in benign and malignant lesions. RESULTS--Vimentin expression was identified on frozen section in the cells of ductal (53%), lobular (86%), and mucinous (33%) carcinomas and in the luminal epithelium of fibroadenomas (68%), cases of fibrocystic disease (47%), and a malignant phyllodes tumour. Formalin fixation reduced the percentage of carcinomas and cases of benign disease in which vimentin was detected. This reduction was more pronounced in fibroadenoma and fibrocystic disease than in ductal carcinoma. Associations were identified between vimentin expression as detected on frozen section and tumour grade, size, number of lymph nodes affected, oestrogen receptor content and growth fraction. Only the association with grade was significant (p = 0.045). There was no significant correlation between any of these prognostic variables and vimentin expression on paraffin wax sections. There was no difference in the intracellular localisation of vimentin staining between benign and malignant lesions, or between low and high grade ductal carcinomas. CONCLUSION--There is some loss of vimentin immunoreactivity after formalin fixation. Vimentin expression does not assist in differentiating between benign and malignant breast disease, but is correlated with tumour grade in ductal carcinoma.     

Use of monoclonal antibody for assessment of estrogen receptor content in fine-needle aspiration biopsy specimen from patients with breast cancer

With increasing emphasis on application of fine-needle aspiration biopsy (FNAB) for an earlier detection of breast cancer, there is clearly a need for a method that requires only a small number of tumor cells for hormone receptor analysis. An immunocytochemical assay utilizing monoclonal antiestrophilin antibody and the peroxidase-antiperoxidase technique has been shown to be highly specific and sensitive for the detection of estrogen receptor (ER) in breast cancer tissues. To assess the usefulness of this technique in FNAB, 62 cases of primary, recurrent, and metastatic breast cancers were studied. The findings from the aspirated cells employing estrogen receptor immunocytochemical assay (ER-ICA) were compared with results obtained from cell population of the same tumors following their removal using both cytochemical (ER-ICA) and biochemical (dextran-coated charcoal assay) methods for ER determination. Overall, there was a 92% concordance between biochemical and cytochemical results. This result suggests that anti-ER monoclonal antibody in immunocytochemical analysis is an effective tool in assessment of ER content in breast cancers. The technique can be easily performed at community hospitals and is well suited for specimens of insufficient size for biochemical assay. It may extend the scope of FNAB and make ER studies possible on material unsuitable for biochemical assay from sites other than breast.