Expression in normal adult, fetal, and neoplastic tissues of a carbohydrate differentiation antigen recognised by antigranulocyte mouse monoclonal antibodies.

The distribution in paraffin fixed human tissues of a carbohydrate antigen defined by two monoclonal antibodies raised against human granulocytes has been studied by means of an immunoperoxidase technique. In addition to granulocytes, the antigen has been detected in adult tissues on identifiable cell types of the stomach, kidney, adrenal medulla, and brain and on the mucins of the gastrointestinal tract and other secretions. In fetal tissue, epithelial cells of the alimentary tract, lung, brain, and kidney express the antigen. Adenocarcinoma of the colon, stomach, breast, and lung are stained strongly, as are other types of lung cancer. The monoclonal antibodies give a staining pattern similar but not identical to other monoclonal antibodies raised against granulocytes or neoplastic cell lines which recognise the antigen 3-fucosyl N-acetyllactosamine. The use of antibodies against this oncofetal antigen in the study of differentiation and as tumour markers is discussed.     

Intracytoplasmic lumina–a useful diagnostic feature of adenocarcinomas

The monoclonal antibody NCRC-11, which has epithelial membrane antigen (EMA)-like immunoreactivity, was used to identify intracytoplasmic lumina in a series of 105 adenocarcinomas from various sites and in 283 breast carcinomas; 55% of the non-breast carcinomas and all breast carcinomas except one of spindle cell type contained intracytoplasmic lumina. The highest frequency (16.4% of tumour cells) was found in invasive lobular carcinoma of the breast. The use of antibodies with EMA reactivity is advocated in the routine investigation of metastatic and undifferentiated tumours.     

Intracytoplasmic lumina--a useful diagnostic feature of adenocarcinomas

The monoclonal antibody NCRC-11, which has epithelial membrane antigen (EMA)-like immunoreactivity, was used to identify intracytoplasmic lumina in a series of 105 adenocarcinomas from various sites and in 283 breast carcinomas; 55% of the non-breast carcinomas and all breast carcinomas except one of spindle cell type contained intracytoplasmic lumina. The highest frequency (16.4% of tumour cells) was found in invasive lobular carcinoma of the breast. The use of antibodies with EMA reactivity is advocated in the routine investigation of metastatic and undifferentiated tumours.     

Epithelial markers in pancreatic carcinoma: immunoperoxidase localisation of DD9, CEA, EMA and CAM 5.2.

Paraffin wax embedded, formalin fixed sections of 22 adenocarcinomas of the exocrine pancreas were stained with four mouse monoclonal antibodies: DD9-E7, an antibody raised against a human pancreatic tumour xenograft; carcino-embryonic antigen (CEA); epithelial membrane antigen (EMA); and cytokeratin (CAM 5.2). An indirect immunoperoxidase technique without enzyme pre-digestion and an affinity-purified sheep anti-mouse peroxidase conjugate were used. All of the tumours were positive for DD9-E7, EMA, and CAM 5.2. Twenty out of 22 were focally positive for CEA and the staining was often weak. As all of these adenocarcinomas were DD9-E7 positive, absence of staining for DD9-E7 in a tumour makes the diagnosis of adenocarcinoma of the exocrine pancreas very unlikely, and this is of value in distinction from endocrine carcinomas with a marked acinar pattern. The weak CEA staining distinguished pancreatic carcinomas from colorectal tumours. Because the distribution of staining for EMA and CAM 5.2 was no different from that previously seen in adenocarcinomas from other sites, these markers are likely to be of limited value in the differential diagnosis of abdominal adenocarcinomas of uncertain origin.     

Occult regional lymph node metastases from breast carcinoma: immunohistological detection with antibodies CAM 5.2 and NCRC-11.

Ninety-eight consecutive patients with primary operable breast cancer and an initial diagnosis of no regional lymph node metastases as assessed by conventional light microscopy were studied. Immunohistological staining of routine lymph node sections was assessed using two monoclonal antibodies: CAM 5.2 (Becton Dickinson) with specificity for low molecular weight cytokeratin, and NCRC-11 (CRC Laboratories, Nottingham) with specificity for epithelial mucin antigen. Positive staining for occult metastases was seen in nine patients with CAM 5.2 and in eight of these nine with NCRC-11. At a follow-up out to 14 years, there was no difference in overall survival, in recurrence-free survival, or in frequency of or time to presentation of local or regional recurrences between occult metastasis-positive and occult metastasis-negative patients. This study concludes that while immunohistological staining of routine lymph node sections increases the diagnostic yield of metastases, it is not to be recommended as this increase is of no useful clinical value.     

Immunological and structural features of the protein core of human polymorphic epithelial mucin.

The protein core of high mol. wt polymorphic epithelial mucin (PEM--approximately 400 kDa glycoprotein) which is associated with breast carcinomas, consists of a repeating 20 amino acid peptide motif [Gendler et al. (1988) J. biol. Chem. 263, 12,820-12,823]. Monoclonal antibodies C595 (anti-urinary mucin) and NCRC-11 (anti-breast carcinoma cells), and other antibodies against human milk fat globule membranes, were found to recognize determinants present within this 20 amino acid peptide. A model of the peptide was developed based on hydropathicity and structure prediction calculations and these indicated that the repeated structure is dominated by a hydrophilic domain of seven amino acids, extending into two flanking beta turns. NMR analysis of the 20 amino acid peptide was undertaken to probe the secondary structure. Epitope mapping experiments involving solid phase synthesis of overlapping heptapeptides in the repeat unit identified the minimum structures for antibody binding as Arg-Pro-Ala-Pro and Arg-Pro-Ala for the C595 and NCRC-11 antibodies, respectively. These determinants were found within the predicted hydrophilic turn region domain of the peptide. The epitopes for six other PEM-reactive monoclonal antibodies were also determined to reside within the predicted hydrophilic turn domain. This evidence is in accord with the disposition of this region of the PEM peptide core being at the exterior of the glycoprotein where it would be accessible to antibody recognition and binding events.     

Keratins versus epithelial membrane antigen in tumor diagnosis: an immunohistochemical comparison of five monoclonal antibodies

Among the monoclonal antibodies capable of detecting epithelial lineage, some recognize keratin and others identify antigens of epithelial membranes. Of the latter, the most commonly used is directed against an antigen present in cell membranes derived from milk fat globules, epithelial membrane antigen (EMA). To determine their relative sensitivity and specificity and hence their diagnostic value, we compared four commercially available monoclonal antibodies to low-molecular-weight keratins--AE1, CAM 5.2, PKK1, and 35 beta H11--with the monoclonal antibody to EMA (anti-EMA). We studied 383 samples of human neoplasms of diverse histogenetic types and degrees of differentiation. Anti-EMA was found to be less sensitive than the monoclonal antibodies to keratin in several epithelial tumors, including tumors of the prostate (11 of 13 negative), gastrointestinal tract (13 of 34 negative), and thymus (seven of eight negative). Anti-EMA was also less sensitive in mesotheliomas (nine of 24 negative). Of the embryonal carcinomas, all stained with the monoclonal antibodies to keratin, whereas none stained with anti-EMA. False-positive staining with anti-EMA was seen in two of 14 T-cell lymphomas. We conclude that the monoclonal antibodies to low-molecular-weight keratins are more sensitive and specific for the identification of epithelial origin of neoplasms than is monoclonal anti-EMA. Anti-EMA should not be used as the sole marker of epithelial differentiation in tumor diagnosis.     

Similarities of antisera to casein and epithelial membrane antigen

Summary Antisera raised against human milk fat globule membranes and against the casein fraction of human milk have been compared. Using an immunohistochemical stain of tissue sections it has been shown that many of the antigenic determinants detected by the different antisera are identical. A radioimmunoassay for epithelial membrane antigen (EMA) showed that casein preparations are associated with small quantities of EMA. Antisera to casein frequently contained appreciable concentrations of antibodies to EMA and this accounts for the immunohistochemical staining of non-mammary tissues.     

Monoclonal antibodies to the human mammary gland

Summary Mouse monoclonal antibodies have been raised to the human milk fat globule membrane. The distribution of the antigens detected by four of the antibodies has been examined in formalin-fixed, paraffin-embedded human tissues by light microscopic immunocytochemistry. The four antibodies stain lactating breast and normal resting breast. Two exclusively stain the luminal membranes of breast epithelial cells. A third antibody stains in addition the lateral membranes of duct epithelial cells. The fourth antibody stains both epithelial and myoepithelial cells. None of the antibodies is breast specific, nor do they stain every epithelial cell within the breast. Instead, each antibody reveals a complex and heterogeneous distribution of staining throughout the normal tissues. Within the breast, the staining by a given antibody is usually segmental and conforms to secretory units and their associated ducts. Similarly heterogeneous patterns of staining are also observed in the extramammary normal tissues. Despite the apparent morphological identity between breast epithelial cells when examined by conventional light microscopy, the hitherto unrecognised functional heterogeneity, which has been revealed by the monoclonal antibodies could have importance in understanding the biology of the normal breast and the pathology of breast cancer.     

Immunocytological staining reactions of anti-carcinoembryonic antigen, Ca, and anti-human milk fat globule monoclonal antibodies on benign and malignant exfoliated mesothelial cells.

A panel of three monoclonal antibodies were used in an immunoalkaline phosphatase staining method on a series of serous effusion samples from cases of mesothelioma, lung carcinoma, and benign disease. The antibodies used were anti-carcinoembryonic (CEA) antigen, Ca, and anti-human milk fat globule membrane antigen. Antibodies to the Ca antigen and human milk fat globule membrane antigen stained 75% and 83% of mesothelioma and 75% of cases of lung carcinoma, respectively. The anti-CEA antibody stained most cases of lung carcinoma strongly but was negative on 11 of 12 cases of mesothelioma and showed weak staining on one case. Benign cases were negative with all three antibodies. These three antibodies may be useful in distinguishing benign and malignant mesothelial cells and lung carcinoma in serous effusions.     

The histogenesis of mammary and extramammary Paget''s disease

The histogenesis of mammary and extramammary Paget's disease has been studied by immunohistochemical staining of paraffin-embedded tissue using a panel of epithelial cell markers, which react with secretory or ductal epithelium, but not stratified epithelium. These markers included a monoclonal antibody E29 to epithelial membrane antigen EMA, the cytokeratin marker CAM 5.2 and three new monoclonal antibodies raised to human milk fat globule membrane (LICR-LON-TW19 and H.10.A) and a human bladder cell cancer line (3.77). The findings demonstrate that both mammary and extramammary Paget's disease are of epithelial cell origin and share antigens expressed by simple epithelia. Some antigens, such as EMA and low molecular weight cytokeratins are consistently present in both diseases, whereas other antigens, identified by H.10.A and TW19 are found more frequently in cases of extramammary Paget's disease. This panel of monoclonal antibodies also proved useful in distinguishing Paget's disease from pagetoid melanoma and clear cell Bowen's disease.     

Immunohistochemical reactivity of a monoclonal antibody prepared against human breast carcinoma

Summary The reactivity profile of an IgM monoclonal antibody, MBR1, raised against the human breast cancer cell line MCF7, was studied in a variety of human tumours and non-neoplastic tissues by light microscopic immunohistochemistry. The range of reactivity included specific types of non-neoplastic epithelial cells and a number of epithelial tumours. Most mammary carcinomas reacted with MBR1, but adenocarcinomas and squamous carcinomas from different sites were also strongly positive. Different patterns of immunoreactivity were apparent in microscopically normal tissues, in tissues with inflammatory changes and in carcinomas. Heterogeneous staining, despite morphological similarities, was documented in neoplastic and non-neoplastic epithelial cells. The reactivity of MBR1 was different from that reported for other monoclonal antibodies, but revealed similarities to that of monoclonal antibodies and polyclonal sera against human milk fat globule membrane.     

JC70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections.

A new monoclonal antibody, JC70, raised against a membrane preparation from a spleen affected by hairy cell leukaemia, recognises a membrane bound glycoprotein identical with that of the CD31 group of monoclonal antibodies. The antibody stains a fixation resistant epitope on endothelial cells in benign and malignant conditions in a wide variety of paraffin wax embedded tissue. JC70 stained malignant endothelial cells in 10 angiosarcomas with more consistency than monoclonal or polyclonal antibodies to factor VIII related antigen (FVIII-Rag). In four cases of Kaposi's sarcoma the antibody stained malignant endothelial cells but not spindle cells. It is concluded that antibody JC70 is of value for studying benign and malignant human vascular disorders in routinely processed tissue.     

The human thymus microenvironment: heterogeneity detected by monoclonal anti-epithelial cell antibodies.

Monoclonal antibodies were raised against human thymus stromal cells and their specificity for the epithelial component of thymus stroma assessed by double immunofluorescence using anti-keratin antibodies to identify epithelium. Our monoclonal antibodies identify six distinct patterns of epithelial cell antigen expression within the thymus: pan epithelial (antibody IP1); cortex (MR3 and MR6); cortical/medullary junction (IP2); subcapsule and subpopulation of medulla (MR10/MR14); Hassall's corpuscles and adjacent subpopulation of medulla (IP3); Hassall's corpuscles only (MR13/IP4). This heterogeneity of antigen expression suggests that many different epithelial microenvironments exist within the human thymus.     

Human monoclonal antibody against human lymphocytic cells. A human monoclonal antibody that reacts preferentially with human lymphocytic cells

Human monoclonal antibodies may replace human or xenogeneic antisera and mouse monoclonal antibodies for therapeutic applications. The human IgM monoclonal antibody Ha6D3 was produced after in vitro immunization and fusion with a mouse myeloma. Its reactivity against human normal and leukemic cells was investigated in cytotoxicity assays, and the antigen distribution in normal tissues was investigated with biotinylated Ha6D3 and avidin-peroxidase complexes. It was shown that Ha6D3 reacts preferentially with human lymphocytic cells. Only moderate reactions with epithelial cells in some organs were observed in cryostat sections, but because of poor accessibility in vivo these reactions were considered to be negligible.     

Epithelial membrane antigen in lymphosarcoma cells (immunomorphologic study)]

11 cases of different types of lymphosarcoma of T- and B-nature are studied immunohistochemically by means of antibodies against epithelial membrane antigen (EMA), including new Soviet monoclonal antibodies ICO-25 to this antigen, common leucocytic antigen, vimentin and cytokeratin. The phenomenon of the EMA including ICO-25 binding to the cells of some lymphosarcomas is confirmed. This indicates that the use of only this "epithelial differentiation marker" in not sufficient for the differentiation between lymphosarcoma and poorly differentiated carcinoma. Variability of lymphosarcoma cells staining by vimentin antibodies is shown, other limitations of the immunohistochemical examination of lymphoma are discussed. The conclusion is made of the necessity to use the spectrum of the above antibodies when differentiating between lymphosarcoma and poorly differentiated carcinoma.     

Critical re-examination of the distribution of aromatase-immunoreactive cells in the quail forebrain using antibodies raised against human placental aromatase and against the recombinant quail, mouse or human enzyme

Mouse and quail aromatase cDNAs were isolated from libraries of mouse ovary and quail brain by using a human aromatase cDNA fragment (hA-24) as a probe. These three cDNAs were inserted into plasmid vectors and expressed in Escherichia coli. Antisera against these purified recombinant proteins were raised in rabbit and purified by ammonium sulfate fractionation and affinity chromatography. The three antibodies directed against recombinant human, mouse and quail proteins were used to visualize aromatase-immunoreactive cells in the quail brain. They were compared with the antibody raised against human placental aromatase used in previous experiments and with another antibody recently developed by similar methods. The signal obtained with all antibodies was completely abolished by preadsorption with the homologous recombinant antigens and the signal produced by the two antibodies raised against placental aromatase was similarly abolished by a preadsorption with recombinant quail aromatase. The antibodies raised against recombinant proteins identified the major groups of aromatase cells previously described in the quail brain. The antibodies directed against the mouse and quail antigen identified more positive cells and stained them more densely than the antibodies raised against human recombinant antigen or purified placental aromatase. The new cell groups identified by the antibody raised against quail recombinant aromatase were located in an area ventral to the fasciculus prosencephali lateralis, the nucleus accumbens, the paleostriatum ventrale, the nucleus taeniae, the area around the nucleus ovoidalis, the caudal tuber and the mesencephalic central gray. A critical re-examination of the distribution and nomenclature of the aromatase-positive cells is proposed based on these new findings.     

Serological and immunochemical characterization of monoclonal antibodies to Toxoplasma gondii.

The fusion of mouse NS-1 myeloma cells with spleen cells from mice chronically infected with Toxoplasma gondii resulted in eight clones of hybridomas producing monospecific antibodies against membrane or cytoplasmic antigens of Toxoplasma tachyzoites. One of the antibodies to a cytoplasmic determinant was an IgM; the others directed to membrane or cytoplasmic antigens belonged to the IgG2 or IgG3 isotypes. Antibodies of clones 1E11 (IgG3), 2G11, and 3E6 (IgG2) directed to membrane antigens, bound complement and were reactive in the complement-dependent cytotoxicity assay of Sabin-Feldman. These IgG2 antibodies were strongly agglutinating to parasites, whereas the IgG3 was relatively weak. Another IgG2 antibody (5B6), possibly recognizing a shared antigen of membrane and cytoplasm, exhibited a low titre in the cytotoxicity assay as well as in the agglutination assay. Two other antibodies to membrane antigens (2B7 and 2F8) as well as an antibody to a cytoplasmic antigen (3G3) did not bind complement and did not cause agglutination. The pattern of parasite staining produced by monoclonal antibodies to membrane antigens in an IFA test was different from that of polyvalent antisera. A strictly localized or 'beaded' staining was observed, as well as a smooth, rim fluorescence. Toxoplasma tachyzoites were surface radio-iodinated and the solubilized membrane proteins were immunoprecipitated with monoclonal antibodies and analysed by two-dimensional polyacrylamide gel electrophoresis. Two independently arising monoclonal antibodies to membrane antigens (2G11 and 3E6) consistently precipitated both the solubilized 35,000 and 14,000 mol. wt proteins, while 1E11 precipitated the 27,000 mol. wt protein.     

Identification of a cycle-modulated 200-kDa endometrial antigen by a monoclonal antibody LDS60.

We have developed a mouse monoclonal antibody, LDS60, against a cycle-dependent antigen by immunising (MF1 x BALB/c)F1 mice with a human endometrial membrane preparation. In formalin fixed paraffin embedded sections, LDS60 identified an epithelial specific antigen which exhibited a specific pattern of expression during the menstrual cycle. It was only occasionally expressed during the proliferative phase. During the early-secretory phase, there was intracytoplasmic staining in about half of the glands examined. This was in the form of small microvesicles, either near the base of the cell or supranuclear. In the mid-secretory phase the same proportion of glands exhibited staining in the form of micro-vesicles that were noted to accumulate nearer to the cell apices. In the late-secretory phase, there was no intracytoplasmic staining and the antigen was localised to the luminal border of the glandular epithelium and some staining appeared within the gland lumen of approximately 20% of glands. It is also diffusely expressed in some mucous secreting cells in the tongue, stomach and colon, as well as lung pneumocytes. The antigen has a molecular weight of approximately 200 kDa as identified by immunoblotting. This antigen exhibits similarities to MUC-1 which is involved in uterine receptivity and could therefore have a similar role. Its cycle modulation suggests that it could be used to monitor the uterine response to steroids.     

Expression of the 17-1A antigen in gastric and gastro-oesophageal junction adenocarcinomas: a potential immunotherapeutic target?

BACKGROUND: A murine monoclonal antibody against the 17-1A epithelial antigen has been shown to be a useful adjuvant therapy in colorectal cancer. Its clinical use could be extended to patients with upper gastrointestinal adenocarcinoma. AIM: To determine the distribution of the antigen in gastric and oesophageal adenocarcinoma. METHODS: The activity of two monoclonal antibodies active against 17-1A epithelial antigen was studied in gastric and gastro-oesophageal junction adenocarcinomas: fresh frozen tissue from both the carcinoma and adjacent mucosa was stained using immunocytochemistry with a murine monoclonal antibody (17-1A edrecolomab, Glaxo Wellcome); paraffin embedded tissue was stained using the humanised monoclonal antibody 3622W94 (Glaxo Wellcome). RESULTS: 29 of 33 cancers (88%) stained with the murine antibody and 39 of 40 (98%) with the humanised antibody. The degree of staining was greater in well differentiated and moderately differentiated tumours. There was no staining of the normal background gastric or oesophageal mucosa, but areas of intestinal metaplasia stained intensely. The humanised monoclonal 3622W94 antibody produced more intense staining than the murine antibody. CONCLUSIONS: The high incidence of expression of the 17-1A antigen in patients with gastric and gastro-oesophageal junction adenocarcinomas suggests a potential role for these antibodies as an adjuvant treatment for these common cancers.