Comparison of immunoperoxidase staining of 3 different types of CD5 antibodies in a spectrum of breast lesions

CONTEXT: We recently described a patient with chronic lymphocytic leukemia who presented with a breast carcinoma that stained positive for CD5 using a commercially available antibody (CD5-4C7, Novocastra, Newcastle upon Tyne, UK). OBJECTIVES: To study the distribution of CD5 immunoreactivity in tissue sections of a variety of benign and malignant breast lesions using the antibody CD5-4C7 and to compare the results with those obtained with 2 other commercially available CD5 antibodies (CD5/54/F6, Dako, Ely, Cambridgeshire, UK, and CD5/54/B4, Novocastra). DESIGN: Paraffin sections of 102 breast biopsy specimens with various diagnoses were examined using the avidin-biotin immunoperoxidase complex technique. SETTING: The histopathology department of a tertiary referral teaching hospital. RESULTS: The staining results obtained with CD5-4C7 were different from those obtained with the other 2 antibodies. With 4C7, the normal and benign biopsy specimens showed varying numbers of positive epithelial cells and lymphocytes. Heterogeneous positive staining was also present in 47 (78%) of 60 invasive female breast carcinomas and in all 3 male breast carcinomas examined. A statistically significant correlation was found between CD5 positivity and tumor grade, with grade 3 tumors being less likely to be CD5 positive than grades 1 and 2 (P =.0035). No correlation was found between CD5 positivity and patient's age, tumor histologic type, axillary lymph node status, or progesterone receptors. On the other hand, the CD5/54/F6 and CD5/54/B4 antibodies only stained lymphocytes and occasional normal breast ducts, mostly those showing apocrine metaplasia. All other normal benign and malignant epithelial cells were negative. CONCLUSIONS: Positive staining for CD5 using the antibody 4C7 is seen in normal and benign breast tissue and 78% of invasive breast carcinomas. The positivity is more common in low-grade tumors. No significant staining was seen with the 2 other CD5 clones used in this study. The significance of the positive staining obtained with CD5-4C7 is not obvious, but this clone may be more sensitive than the others, or it may be recognizing an epitope shared by another antigen.     

An immunocytochemical method for demonstrating estrogen receptor in human uterus using monoclonal antibodies to human estrophilin

We have used monoclonal antiestrophilin antibodies to develop an improved immunocytochemical method for localizing estrogen receptors in tissue sections with an indirect immunoperoxidase technique. Rat monoclonal antibodies were raised against human estrogen receptor (estrophilin) protein derived from the cytosol of MCF-7 human breast cancer cells. These monoclonal antibodies have been shown to have extensive cross-reactivity with estrogen receptors from various primate and nonprimate tissues. We have used frozen sections of human proliferative phase endometrium and an indirect immunoperoxidase technique to establish conditions for demonstrating estrogen receptor antigenic determinants in frozen tissue sections. The method involves (a) brief fixation with formaldehyde-containing fixatives either prior to freezing or immediately after cutting cryostat sections, (b) bleaching the tissue of endogenous peroxidase activity, (c) application of primary antibody or control immunoglobulin, (d) application of an adsorbed bridging antibody (goat antirat IgG), and (e) application of rat peroxidase-antiperoxidase followed by diaminobenzidine. Specific nuclear staining for estrogen receptor antigenic determinants was observed in the vast majority of epithelial and stromal cells. No specific cytoplasmic staining was identified in cryostat sections of any of the 17 cases studied for this report.     

ME1. A monoclonal antibody that distinguishes epithelial-type malignant mesothelioma from pulmonary adenocarcinoma and extrapulmonary malignancies.

ME1 is a monoclonal antibody reactive in frozen tissue sections with normal mesothelial cells and malignant mesotheliomas. In this immunoperoxidase study, ME1 reacted with all 40 epithelial type malignant mesotheliomas. Fifty percent or more of the mesothelioma cells were stained in all cases and the staining intensity was strong in 32 and moderate in eight. In contrast, all 19 well- and moderately differentiated pulmonary adenocarcinomas were completely negative, and of the total 88 non-mesotheliomatous malignancies studied, staining comparable to the mesotheliomas was seen in only 6 tumors (2 pulmonary adenocarcinomas, 2 adenocarcinomas of the breast, 1 adenocarcinoma of the pancreas, and 1 melanoma), although limited, weaker staining was seen in additional cases. Five of the six strongly to moderately positive nonmesotheliomatous tumors had immunoreactivity for complementary immunoreactants (CEA, Leu-M1, S-100 protein, HMB-45). Our results with ME1, the first monoclonal antibody that recognizes malignant mesothelial cells, provides a basis for using this reagent in the differential diagnosis of tumors of the pleura and peritoneum.     

Survival in breast cancer related to tumour oestrogen receptor status and immunohistochemical staining for NCRC 11

NCRC 11 is a monoclonal antibody raised against dissociated breast cancer cells. Using this antibody, it has been shown in an earlier study that staining of a high proportion of tumour cells is strongly associated with superior survival. Many investigators have found that positive tumour oestrogen receptor (OER) status is associated with better survival in breast cancer. In the present study of 136 breast cancer patients followed up for a minimum of 30 months, tumour oestrogen receptor assays were carried out, and using the indirect immunoperoxidase technique, histological sections of paraffin embedded material were stained for NCRC 11; for the purpose of this study, tumours showing 25 per cent or less cells staining were regarded as 'negative'. Patients whose tumours were OER positive had a significantly better prognosis (logrank test P less than 0.05). Patients whose tumours were graded as negative for NCRC 11 had a poorer prognosis compared with the positive group but this did not attain significance. Our failure to demonstrate convincingly a better prognosis for the NCRC 11 positive patients may relate to the shorter duration of our study, or to different tissue fixation techniques. In this study staining for NCRC 11 was positively correlated with oestrogen receptor status (P less than 0.02).     

Cross-reactivity with normal antigens in commercial anti-CEA sera, used for immunohistology. The need for tissue controls and absorptions

The demonstration of carcinoembryonic antigen (CEA) in serum and in tissue sections may be of value in diagnosis and follow-up of several malignant tumors. Anti-CEA sera, however, cross-react with normal antigens, also present in tumors. The staining patterns of three commercially produced anti-CEA sera were investigated on sections of benign and malignant tissues, using an indirect immunoperoxidase technic. All three anti-CEA sera tested showed cross-reactions. A rapid, simple, and reproducible immunoabsorption was developed. After absorption, the reactivity of the sera with normal colon mucosa and carcinomas of the colon remained positive, whereas sections of benign tissues were negative. Moreover, four of ten breast cancers, positive with unabsorbed antisera, became negative after absorption. These findings stress the importance of immunohistochemical negative and positive controls. The use of well-absorbed commercial anti-CEA sera can lead to more uniform results, exchangeability of results, and a better understanding of the value of this tumor marker.     

Quantitative assessment of Tn antigen in breast tissue micro-arrays using CdSe aqueous quantum dots

In this study, we examined the use of CdSe aqueous quantum dots (AQDs) each conjugated to three streptavidin as a fluorescent label to image Tn antigen expression in various breast tissues via a sandwich staining procedure where the primary monoclonal anti-Tn antibody was bound to the Tn antigen on the tissue, a biotin-labeled secondary antibody was bound to the primary anti-Tn antibody, and finally the streptavidin-conjugated AQDs were bound to the biotin on the secondary antibody. We evaluated the AQD staining of Tn antigen on tissue microarrays consisting of 395 cores from 115 cases including three tumor cores and one normal-tissue core from each breast cancer case and three tumor cores from each benign case. The results indicated AQD-Tn staining was positive in more than 90% of the cells in the cancer cores but not the cells in the normal-tissue cores and the benign tumor cores. As a result, AQD-Tn staining exhibited 95% sensitivity and 90% specificity in differentiating breast cancer against normal breast tissues and benign breast conditions. These results were better than the 90% sensitivity and 80% specificity exhibited by the corresponding horse radish peroxidase (HRP) staining using the same antibodies on the same tissues and those of previous studies that used different fluorescent labels to image Tn antigen. In addition to sensitivity and specificity, the current AQD-Tn staining with a definitive threshold was quantitative.     

Immunohistochemical localization of epidermal and Mallory body cytokeratin in undifferentiated epithelial tumors. Comparison with ultrastructural features

Twenty-one anaplastic tumors were studied by light microscopy (LM), immunoperoxidase staining using anti-epidermal cytokeratin (ECK) and anti-Mallory body cytokeratin (MBCK) antibodies, and electron microscopy (EM), to determine whether an epithelial origin could be confirmed. The tumors were derived from lung, stomach, colon, breast, uterus, kidney, bladder, and mesothelium. By LM, the tumors consisted of either large and polygonal, spindle or small, round cells. With immunoperoxidase staining, 11 (52%) of the anaplastic tumors were positive for ECK, positivity being either absent or only weak in the main tumor mass, but marked in areas of infiltration and metastases. In contrast, all of the anaplastic tumors were positive for MBCK in the main tumor mass, infiltrating areas, and metastases. In the case of adenocarcinomas, staining was either web-like or diffuse throughout the cytoplasm with concentration occurring at the cell surface, whereas in mesotheliomas, the staining was either diffuse or showed focal perinuclear accentuation. Twelve of 13 anaplastic tumors examined by EM showed epithelial features (desmosomes, tonofilaments, lumina, and/or microvilli). As controls, 21 non-epithelial tumors (five melanomas, eight sarcomas, and eight lymphomas) showed no reactivity with either cytokeratin antibody. These studies show that the epithelial nature of undifferentiated and poorly differentiated tumors can be confirmed by immunohistochemistry using anti-cytokeratin antibodies.     

Estimation of hormone receptor status in fine-needle aspirates and paraffin-embedded sections from breast cancer using the novel rabbit monoclonal antibodies SP1 and SP2

We describe a method of immunocytochemical assessment of estrogen receptor (ER) status on alcohol-fixed smears obtained by fine-needle aspiration (FNA) from breast cancer patients, using a commercially available rabbit monoclonal antibody anti-ER (SP1) without any antigen retrieval. A series of 40 aspirates were analyzed and the results of ER status were compared with the respective formalin-fixed tissue using the same procedure and with assessment by the classical method using the mouse monoclonal antibody 6F11 (anti-ER) with antigen retrieval on paraffin sections. Twenty-four out of the 40 cases examined were positive at least by two methods and 16 were negative for all three determinations. The results obtained in the ER immunocytochemical assay on aspirates and paraffin sections using the antibody SP1 and those obtained on paraffin sections using the antibody 6F11 were quite similar. In one case the material was insufficient to interpret the reaction in the cytological specimen and only one case, with focal positivity reaction on paraffin sections, was negative in the cytological specimen. The intensity of nuclei staining in cytological smears of breast cancer cells was stronger than that observed by traditional methods. We also assessed progesterone receptor (PR) status on 40 paraffin-sections from breast cancer patients, using a commercially available rabbit monoclonal antibody anti-PR (SP2), with the same characteristics described for anti-ER (SP1). The results were compared with assessment by the classic method with mouse monoclonal antibody 1A6 (PR) on paraffin sections and total agreement was observed. Of the 40 cases examined, 18 were positive and 22 were negative for the two determinations. We conclude that the application of the ER method on alcohol-fixed smears/paraffin sections with the rabbit monoclonal antibody SP1, and the PR method on paraffin sections with the rabbit monoclonal antibody SP2, provide several advantages, such as high sensitivity and specificity of the reaction, stronger immunostaining, shorter procedures times, and avoidance of antigenic retrieval methods.     

Quantification of oestrogen receptors in breast cancer: radiochemical assay on cytosols and cryostat sections compared with semiquantitative immunocytochemical analysis.

A radiochemical oestrogen receptor assay on cytosol was correlated with a radiochemical and an immunohistochemical oestrogen receptor assay using cryostat sections from 50 breast cancer specimens. Oestrogen receptors were reliably quantitated in 6 micron cryostat sections with Scatchard analysis using radiolabelled oestradiol, and a good quantitative and qualitative relation with cytosol oestrogen receptor assay was found. Parallel sections were used for routine histological tissue verification and for direct comparison with immunohistochemistry for oestrogen receptor. Specific immunoperoxidase staining with a rat monoclonal antibody was scored by semiquantitative evaluation of the staining intensity of cancer cell nuclei. Oestrogen receptor scoring was highly reproducible when performed by the same observer. The semiquantitative immunohistochemical oestrogen receptor score correlated significantly better with the radiochemical assay on sections than with cytosol assay. Oestrogen receptor in breast cancer can be reliably assayed by semiquantitative evaluation of cryostat sections immunostained for oestrogen receptor, but only if the procedure is adequately standardised. The results underline the importance of cellular heterogeneity as a cause of variation in oestrogen receptor assay evaluation in breast cancer.