Expression of a new mucin-type glycoprotein in select epithelial dysplasias and neoplasms detected immunocytochemically with Mab A-80

We studied by immunocytochemistry 573 tissue and 106 cytologic samples of human tumors, non-neoplastic proliferative lesions and normal tissues with the monoclonal antibody (Mab) A-80 that recognizes a mucinous glycoprotein from the colon carcinoma cell line LS-174T. The spectrum of benign and malignant breast lesions was studied as were epithelial tumors of the colon, stomach, pancreas, lung, salivary glands, thyroid, prostate, kidney, endometrium, skin and mesothelium; non-epithelial tumors included lymphomas, melanomas, gliomas, meningiomas, and sarcomas of soft tissue and bone. With a single exception, breast carcinomas regardless of histologic type were reactive while few fibroadenomas stained weakly and focally. In fibrocystic disease, the presence and intensity of the reactivity paralleled the severity of the epithelial proliferation, e.g. staining was strong in foci of severe or atypical hyperplasia, borderline lesions and carcinomas in situ; apocrine metaplasia stained often but less strongly. Barrett's mucosa, colonic polyps and most gastric and colonic carcinomas stained regardless of glandular features while small cell neuroendocrine carcinomas did not. Adenocarcinomas of the pancreas and lung, and a subset of large cell lung carcinomas reacted whereas neuroendocrine carcinomas of those sites did not. Carcinomas of endometrium, ovary and prostate reacted variably whereas thyroid and renal carcinomas and mesotheliomas were either negative or weakly reactive despite the presence of glands. Lymphomas, skin adnexal tumors, nevi, schwannomas, melanomas, gliomas and sarcomas generally did not react but occasional A-80-positive cells were seen in rare sarcomas and meningiomas. Immunostaining patterns in cytologic specimens were similar to the aforementioned. We conclude that Mab A-80 is an excellent marker for breast carcinomas, and for certain proliferative forms of fibrocystic disease that may precede or be associated with carcinomatous transformation. In colonic, pulmonary and gastric carcinomas, staining with Mab A-80 revealed exocrine features regardless of the absence of glands whereas in renal and thyroid carcinomas and in mesotheliomas staining was focal and weak or absent irrespective of glandular features. We suggest that Mab A-80 is a very promising immunolabel for select exocrine carcinomas, and for some of the dysplasias that may precede their development; its ease of application on tissue sections and cytologic specimens should broaden its usefulness.     

Immunocytochemical evaluation of neoplastic and non-neoplastic breast diseases with Mab A-80

Five hundred breast tissue samples from 404 cases were immunostained with A-80, a murine IgM Mab that recognizes a mucinous glycoprotein associated with exocrine differentiation. Samples included 196 primary breast carcinomas, 30 breast carcinoma metastases, 118 fibrocystic disease (FCD), and a further group of 84 samples of FCD from cases known to have breast carcinoma. These samples represented a broad spectrum of common and rare variants of carcinoma and FCD. Samples of fibroadenomas, lactating adenomas, cystosarcoma phylloides, gynecomastia, and normal breasts were similarly studied. The vast majority of carcinomas, 203/212 (95.7%) were immunoreactive; staining varied in extent and intensity, and was virtually unrelated to histologic type and to the presence or absence of recognizable glands. In samples including in-situ and infiltrating ductal or lobular carcinoma, reactivity was frequently stronger in the infiltrating components. No significant difference in reactivity between primary and metastatic carcinomas was noted. Of the group of 118 FCD, 27 were negative whereas 91 showed focal and weak staining. Seventy-two/84 FCD with associated carcinoma were immunostained; in 13 of those 72, staining was strong and extensive. Fibroadenomas, lactating adenomas, gynecomastia, and normal "resting" and lactating breast samples stained focally or not at all. Our findings indicate that Mab A-80 is an excellent immunohistochemical marker for the overwhelming majority of breast carcinomas whereas it marks weakly or not at all the majority of benign neoplasms and normal breast. Moreover, Mab A-80 recognizes a subset of FCD that includes proliferative variants associated with an increased incidence of carcinoma, and FCD in association with carcinoma. Questions regarding rare breast carcinomas that do not react with Mab A-80 remain unclear; yet, we believe that Mab A-80 is a highly promising marker of malignant and dysplastic breast epithelium.     

Characterization of a monoclonal antibody against a mucin-type glycoprotein in human sweat

We report the preparation and characterization of an IgG2 monoclonal antibody (MAb), HSMA, prepared against a human pooled sweat extract (HPSE). The major component of HPSE was a mucin-type molecule, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with periodic acid-Schiff (PAS) reagent. By immunoblotting, HSMA revealed a smear in the high molecular weight range, typical of mucins. In enzyme-linked immunosorbent assay (ELISA), HSMA failed to react with HPSE fractions isolated after anionic exchange gel chromatography. Similarly, radio-immunobinding assays demonstrated no reactivity between HSMA and A, B, H, and Lewis blood group-related structures. The immunohistological labeling on normal skin showed that HSMA reacted with the cells of eccrine sweat glands, and to a lesser extent, with sebaceous glands and epidermal cells. Periodate treatment in situ abolished these reactions, thus suggesting the carbohydrate structure of the HSMA-epitope. In indirect immunofluorescence (IF) studies, HSMA also reacted with other exocrine glands, e.g. mammary glands, sublingual glands, mixed sero-mucous glands of the trachea, and in the pancreas. Sparse positive cells were also observed in the testis, kidney, thyroid and digestive tract.     

Demonstrability of the glycoprotein A-80 in postradiation prostatic carcinoma

Radiation therapy results in significant morphological changes in prostatic carcinoma, including decreased cancer size, acinar shrinkage and distortion, cytoplasmic vacuolization, and nuclear pyknosis. Benign acini usually display enlarged, atypical cells with hyperchromatic nuclei. These changes confound the evaluation of limited postradiation samples. The glycoprotein A-80 is known to be upregulated in prostatic intraepithelial neoplasia (PIN) and prostatic carcinoma. In this study, we assessed the expression of A-80 in radiation-treated prostatic carcinoma. Paraffin sections from 64 postradiation prostatic carcinomas obtained at salvage prostatectomy were immunostained with a monoclonal antibody to A-80; selected sections were doubly immunostained with antibodies to A-80 and various cytokeratin polypeptides. All cases showed readily detectable and often intense staining in the cytoplasm of cancer cells and in intraluminal material of malignant acini. The extent and intensity of the reactions were independent of cancer size and grade. Strong reactions were seen in preserved and distorted acini, clear cell areas, single cancer cells, and in colloid pools with few or no recognizable cancer cells. PIN was present in 34 cases (53%), of which 27 (79%) stained strongly for A-80; atrophic and hyperplastic acini generally did not stain, irrespective of the degree of cellular atypia. The A-80 glycoprotein appears remarkably durable and is readily demonstrable in postradiation prostatic carcinoma despite profound architectural and cytologic changes. This characteristic may prove useful in evaluating small samples for confirmation of diagnosis and determination of extent of residual or recurrent prostatic carcinoma after radiation therapy.     

Demonstrability of the glycoprotein A-80 in postradlation prostatic carcinoma

Radiation therapy results in significant morphological changes in prostatic carcinoma, including decreased cancer size, acinar shrinkage and distortion, cytoplasmic vacuolization, and nuclear pyknosis. Benign acini usually display enlarged, atypical cells with hyperchromatic nuclei. These changes confound the evaluation of limited postradiation samples. The glycoprotein A-80 is known to be upregulated in prostatic intraepithelial neoplasia (PIN) and prostatic carcinoma. In this study, we assessed the expression of A-80 in radiation-treated prostatic carcinoma. Paraffin sections from 64 postradiation prostatic carcinomas obtained at salvage prostatectomy were immunostained with a monoclonal antibody to 3–80; selected sections were doubly immunostained with antibodies to A-80 and various cytokeratin polypeptides. All cases showed readily detectable and often intense staining in the cytoplasm of cancer cells and in intraluminal material of malignant acini. The extent and intensity of the reactions were independent of cancer size and grade. Strong reactions were seen in preserved and distorted acini, clear cell areas, single cancer cells, and in colloid pools with few or no recognizable cancer cells. PIN was present in 34 cases (53%), of which 27 (79%) stained strongly for A-80; atrophic and hyperplastic acini generally did not stain, irrespective of the degree of cellular atypia. The A-80 glycoprotein appears remarkably durable and is readily demonstrable in postradiation prostatic carcinoma despite profound architectural and cytologic changes. This characteristic may prove useful in evaluating small samples for confirmation of diagnosis and determination of extent of residual or recurrent prostatic carcinoma after radiation therapy.     

Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors

The mucin-type glycoprotein podoplanin is specifically expressed by lymphatic but not blood vascular endothelial cells in culture and in tumor-associated lymphangiogenesis, and podoplanin deficiency results in congenital lymphedema and impaired lymphatic vascular patterning. However, research into the biological importance of podoplanin has been hampered by the lack of a generally available antibody against the human protein, and its expression in normal tissues and in human malignancies has remained unclear. We generated a human podoplanin-Fc fusion protein and found that the commercially available mouse monoclonal antibody D2-40 specifically recognized human podoplanin, as assessed by enzyme-linked immunosorbent assay and Western blot analyses. We found that, in addition to lymphatic endothelium, podoplanin was also expressed by peritoneal mesothelial cells, osteocytes, glandular myoepithelial cells, ependymal cells, and by stromal reticular cells and follicular dendritic cells of lymphoid organs. These findings were confirmed in normal mouse tissues with anti-podoplanin antibody 8.1.1. Podoplanin was also strongly expressed by granulosa cells in normal ovarian follicles, and by ovarian dysgerminomas and granulosa cell tumors. Although podoplanin was primarily absent from normal human epidermis, its expression was strongly induced in 22 of 28 squamous cell carcinomas studied. These findings suggest a potential role of podoplanin in tumor progression, and they also identify the first commercially available antibody for the specific staining of a defined lymphatic marker in archival human tissue sections, thereby enabling more widespread studies of tumor lymphangiogenesis in human cancers.     

Cell localization of mucin-type receptors assayed with novel GalNac/Gal-specific lectin from sea musselCrenomytilus grayanus in human colon tumors

Cell localization of mucin type receptors in low-differentiated human colon adenocarcinoma was studied. Several types of receptor localization were identified. It was shown that lectin CGL most intensively binds to intracellular membranes, cytoplasm, and secrete of mucin-producing tumor cells. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 10, pp. 441–444, October, 1999     

Expression of the cell-cell adhesion glycoprotein cell-CAM 120/80 in normal human tissues and tumors.

Polyclonal and monoclonal antibodies raised to the 80 kd glycoprotein component of the cell to cell adhesion molecule cell-CAM 120/80 were used to map its distribution immunohistochemically in normal human tissues and in benign and malignant tumors. Cell-CAM 120/80 was found in all normal epithelial tissues, but was not expressed on neural, lymphoid, smooth, striated and cardiac muscle, connective tissue, or the germ cells in either sex. The expression of this adhesion molecule was polarized in ductal and glandular epithelia and evenly circumferential in squamous and transitional epithelia. Some organs, such as the kidney, liver and endocrine glands, showed unique organ to tissue specific patterns. Maturation-dependent loss of cell-CAM 120/80 was noticed in superficial layers of squamous epithelium and the placenta. Benign epithelial tumors expressed cell-CAM 120/80 in a manner comparable with their tissue of origin. Malignant tumors expressed cell-CAM 120/80 either in a manner similar to the tissue of their origin or assumed a less polarized phenotype. Overall, the immunoreactivity in many malignant tumors appeared weaker and the polarization was less pronounced. Thus, cell-CAM 120/80 is a universal marker of human epithelial cells, but its mode of expression differs in various anatomic sites, and may be influenced by maturation or malignant transformation of cells.     

Expression of a high molecular weight mucin-type glycoprotein in human colon cancer as defined by monoclonal antibody 1D3

Murine monoclonal antibody 1D3 recognizes a high molecular weight acidic mucin restricted to the epithelium of normal colonic mucosa and goblet cells. Of 72 colonic carcinoma specimens examined, 29 were found to have detectable level of 1D3 antigen by an indirect immunoperoxidase staining assay on fixed, paraffin-embedded tissue sections. In some specimens a focal staining pattern was observed, while in others 50-90% of tumor cells were stained. Of 28 cases having adjacent normal mucosa, all 28 showed intense staining reaction in the normal mucosa and goblet cells despite the fact that 18 of the tumors were unstained. One of 1 colonic diverticulosis, 2 of 2 ulcerative colitis, 3 of 3 villo-glandular polyps, 19 of 20 adenomatous polyps and 17 of 19 hyperplastic polyps were also stained heavily for the 1D3 antigen. Colonic carcinomas displayed a range of staining patterns and a great degree of antigenic heterogeneity. Well-differentiated tumors characterized by typical goblet cells were almost always positive (10 of 12). As cellular structure became disorganized, as in moderately-differentiated tumors, about 33% of the tumors (17 of 51) stained for 1D3 antigen. As the tumor became more invasive with further disorientation of cellular features, as in poorly differentiated tumors, very few specimens (2 of 9) were positive. It was apparent that with the progression of de-differentiation there was a gradual loss of 1D3 antigen in human colonic tumors.     

An immunohistochemical study of neovasculature in human brain tumors

In order to elucidate the characteristics of newly-formed small blood vessels in brain tumors, 51 cases of various types of brain tumor were studied immunohistochemically. Endothelial cells in all tumor types reacted positively with anti-Factor VIII-related antigen (F-VIII Ag) and UEA-1. Reactivity to UEA-1 was limited to endothelial cytoplasm. Proliferating immature endothelial cells showed weak reactivity to anti-F-VIII Ag. In some cases of benign tumors diffuse reactivity to anti-F-VIII Ag was noted throughout thickened medial areas of the vascular walls, in which reactivity to anti-fibrous collagen and anti-fibronectin was noted. UEA-1 was thought to be a more specific endothelial marker than F-VIII Ag, especially for proliferating and immature endothelial cells. It is strongly suspected that fibrous collagens are related to the formation of a thickened vascular wall in newly-formed small tumor vessels.     

The demonstration of immunohistochemical biomarkers in methyl methacrylate-embedded plucked human hair follicles

Plucked human hair follicles have been proposed as a potential surrogate for tumour tissue for measuring the effect of drugs on pharmacodynamic biomarkers in drug intervention studies. We describe a new technique of embedding plucked hair follicles in the acrylic resin, methyl methacrylate, and the immunohistochemical demonstration of six potential biomarkers (Ki67, EGFR, phospho-p27, phospho-histone H3, phospho-MAPK and phospho-Rb) in de-plasticised sections. The advantages of this technique over those that have been used in support of clinical drug trials, such as skin and tumour biopsies, whole blood and whole hair samples is discussed.     

Usefulness of a novel monoclonal antibody against human osteocalcin in immunohistochemical diagnosis

Summary A novel monoclonal antibody against human osteocalcin, recently established in our laboratory, was shown by immunoblotting and immunohistochemistry to react specifically with human osteoblasts. In the present study, the antibody was applied to the immunohistochemical diagnosis of human bone tumours, especially osteoblastic tumours. The antibody reacted with all 27 osteosarcomas. No positive reaction was found either in chondrosarcoma, giant cell tumours of bone, soft tissue tumours or epithelial tumours. A positive reaction was found preferentially in the cytoplasm of most of the osteosarcoma cells, but not in the extracellular matrix. Since the antibody reacted with formalin-fixed and paraffin-embedded tissues, it will be a useful tool for routine immunohistochemical diagnosis of osteoblastic lesions.     

5T4 Oncotrophoblast Glycoprotein： Janus Molecule in Life and a Novel Potential Target against Tumors

5T4 oncotrophoblast glycoprotein is a transmembrane protein expressed on the embryonic tissue and various malignant tumor cell surfaces. It plays a vital role in the multiple biological and pathological processes including massive cellular migration during the embryogenesis, cell invasion associated with implantation, and neoplastic metastasis in the progression of tumorigenesis. Its restricted profile of expression stratifies criteria of tumorassociated antigen and makes it a new promising candidate for immunotherapy for cancer. Hence, illustrating this molecular function is necessary for discovering the principle of the tumor diffusion and aggravation and is helpful for developing novel and effective strategies of cancer therapy.     

Semaphorin3A immunohistochemical expression in human meningiomas: correlation with the microvessel density

The immunoexpression of the antiangiogenic factor semaphorin3A (SEMA3A) was evaluated in a series of meningiomas. Then, its correlations with the microvessel density (MVD) of the tumors and with the clinicopathological parameters as well with the survival time or recurrence-free interval were investigated. A positive SEMA3A immunostaining was found in most of meningiomas and a significant association was found between a high expression of this protein and a low MVD of the tumors. Moreover, a low SEMA3A immunoexpression was significantly correlated with a higher recurrence rate of meningiomas. In conclusion, our findings suggest a role for SEMA3A as an antiangiogenic factor in meningiomas with its decrease being associated with the development of recurrences. The supplementation of SEMA3A might be used in novel therapeutic antiangiogenic strategies to prevent the recurrence of highly vascularized meningiomas. Dedicated to my son Alessandro who was born on the 3rd of October 2008.     

GABA neurons in the cat red nucleus: a biochemical and immunohistochemical demonstration

Following unilateral kainic acid lesioning of neuronal cell bodies in the cat red nucleus (RN), a large decrease in glutamic acid decarboxylase (GAD) activity was detected in the injected RN, compared to the RN from control, non-injected animals. Using GAD immunohistochemistry, reactive perikarya were visualized dorsolaterally to the rostral part of the nucleus as well as within the RN proper. Taken together, these results point to the existence of an intrinsic GABAergic innervation in the RN area of the cat. The GABAergic neurons characterized here might thus correspond to the inhibitory interneurons previously detected electrophysiologically as a putative source of GABA for large-sized neurons of the RN.