Treatment with mANT2 shRNA enhances antitumor therapeutic effects induced by MUC1 DNA vaccination.

In this study, we developed a combination therapy (pcDNA3/hMUC1+mANT2 shRNA) to enhance the efficiency of MUC1 DNA vaccination by combining it with mANT2 short hairpin RNA (shRNA) treatment in immunocompetent mice. mANT2 shRNA treatment alone increased the apoptosis of BMF cells (B16F1 murine melanoma cell line coexpressing an MUC1 and Fluc gene) and rendered BMF tumor cells more susceptible to lysis by MUC1-associated CD8(+) T cells. Furthermore, combined therapy enhanced MUC1 associated T-cell immune response and antitumor effects, and resulted in a higher cure rate than either treatment alone (pcDNA3/hMUC1 or mANT2 shRNA therapy alone). Human MUC1 (hMUC1)-loaded CD11c(+) cells in the draining lymph nodes of BMF-bearing mice treated with the combined treatment were found to be most effective at generating hMUC1-associated CD8(+)IFNγ(+) T cells. Furthermore, the in vitro killing activities of hMUC1-associated cytotoxic T cells (CTLs) in the combined therapy were greater than in the respective monotherapies. Cured animals treated with the combined treatment rejected a rechallenge by BMF cells, but not a rechallenge by B16F1-Fluc cells at 14 days after treatment, and showed MUC1 antigen-associated immune responses. These results suggest that combined therapy enhances antitumor activity, and that it offers an effective antitumor strategy for treating melanoma.

Thrombin-antithrombin III and D-dimer plasma levels in patients with benign or malignant ovarian tumours.

In 50 patients with benign ovarian tumours, 39 malignant ovarian carcinoma patients and 39 age-matched healthy women, plasma levels of thrombin-antithrombin III complex and D-dimer were determined as well as CA 125. The coagulation activation marker thrombin-antithrombin III complex and D-dimer levels were elevated in the malignant group compared to the benign and control groups. The results suggest that coagulation and fibrinolysis must play a prominent role in ovarian cancer. Moreover, D-dimer and thrombin-antithrombin III were equally useful as CA 125 for the discrimination of patients with benign or malignant ovarian tumours as evidenced by receiver operating and likelihood ratio calculations.

[The expression of proliferating cell nuclear antigen in breast phylloides cystosarcoma and its significance].

OBJECTIVE: To investigate the expression of proliferating cell nuclear antigen (PCNA) in breast phylloides cystosarcoma and its clinicopathological signification. METHODS: Immunohistochemistry (SP method) with monoclonal antibodies PC10 against PCNA was performed in 100 cases of phylloides cystosarcoma and 39 cases of adenofibroma. RESULTS: The positive rate of PCNA in phylloides cystosarcoma was 86%. The average PCNA index (PI) of phylloides cystosarcoma was significantly different among every histologic grading (F = 85.33, P < 0.01). The degree of differentiation was lower, the PI was higher. The PI was closely correlated with histologic grading (r's = 0.77). The PI in grade I phylloides cystosarcoma was higher than that in the group of adenofibroma with abundant mesenchymal cells (t = 3.42, P < 0.01). There was only a low correlation between PI and mitotic figures in phylloides cystosarcoma (r = 0.39). CONCLUSIONS: These findings suggest that the detection of PCNA has considerable practical value in reflecting the proliferation of activity of phylloides cystosarcoma, assisting the pathologists to make histologic grading; distinguishing the malignancy from benign ones (differential diagnosis) and evaluating the prognosis.

Tissue polypeptide antigen immunostaining in fine needle aspirates from solid breast nodules.

The purpose of this study was to determine the role of Tissue Polypeptide Antigen (TPA) immunostaining in the evaluation of aspiration smears from solid breast nodules and to discuss its possible clinical applications. Seventy-one cytologic specimens were studied; 52 were carcinomas, 15 fibroadenomas and 4 fibrocystic dysplasia. TPA was visualized with the peroxidase-antiperoxidase method, using 3-3' diaminobenzidine as chromogen and hematoxylin as counterstaining. The incidence of TPA-positive aspiration smears was 88.5% in the carcinomas and 26.7% in the fibroadenomas. The four cases of fibrocystic dysplasia showed variable behaviour. The intensity of the TPA staining varied from - to and was most frequently high in the carcinomas. The results of this research show a good correlation between a diagnosis of carcinoma and TPA positivity in fine needle aspirates of solid breast nodules.

Mucin-like carcinoma-associated antigen (MCA) in tissue and serum of patients with breast cancer: clinical applications in prognosis and disease monitoring.

Mucin-like Carcinoma-associated Antigen (MCA) has been associated with many breast cancers. The aim of this study was to evaluate MCA in tumor tissue and serum as a potential tumor marker for prognosis and disease monitoring. MCA levels were determined in the tissue of 196 patients with primary breast cancer, 25 with metastatic disease and 25 patients with benign diseases, in pellet and/or cytosol. MCA levels were also determined in the serum of 50 patients with benign diseases, 148 with primary breast cancer (Mo), 150 with metastatic breast cancer (MT), and 200 with no clinical evidence of disease (NED). MCA tissue concentrations in pellet and cytosol were similar: 66.7 + 251 U/mg and 41.1 + 53 U/mg, respectively. No relationship between MCA levels and tumor size or nodal involvement was found. Higher MCA levels were observed in patients with ER+ or PgR+ tumors than in those with ER- or PgR- tumors (p < 0.01). Patients with MCA pellet concentrations lower than 10 U/mg of protein had shorter disease-free intervals (DFI) than those with higher values (p < 0.05). Abnormally high serum levels of MCA were found in 8% of patients with benign diseases, 4% of NED patients, 22% of Mo patients, and in 76% of MT cases. In primary breast cancer MCA values were related to tumor size and nodal involvement. A trend toward a lower DFI in patients with elevated presurgical MCA levels was observed but was of no statistical significance. These differences became statistically significant when patients were subdivided according to nodal status, with shorter DFI in those without nodal invasion (p < 0.05). In metastatic patients, changes in serum MCA were related to the tumor's response to treatment in 82% of cases. The highest MCA values were found in patients with liver or bone metastasis and the lowest values were found in those with locoregional recurrence. In conclusion, although MCA is not a specific tumor marker, it can be useful as a prognostic factor (tissue and serum) and in monitoring metastatic patients.

Mucin-like carcinoma-associated antigen (MCA) in breast cancer: clinical experience at the National Cancer Institute of Milan.

Mucin-like Carcinoma-associated Antigen (MCA) is a glycoprotein belonging to the mucin family; it is defined by the monoclonal antibody b-12. Mucins represent an interesting group of tumor markers and are widely utilized in the clinical monitoring of neoplastic patients. These molecules show a certain degree of tissue specificity and MCA is preferentially associated with breast tissue. Several studies have demonstrated that patients with breast cancer usually have high MCA serum levels. In this paper the experience of the National Cancer Institute of Milan with the clinical use of MCA in breast cancer patients is reported. The observed sensitivity of the MCA test was poor in patients with early-stage disease, while it was acceptable in patients with advanced breast cancer. MCA concentrations appeared to be directly related to disease spread. A clear relationship was seen between MCA levels and lymph-nodal status. The highest MCA plasma levels were observed in patients with metastatic disease. In this group of patients the sensitivity of the test on the basis of a cut-off of 11 U/mL was 52%.

Immunocytochemical reactivity of a mouse monoclonal antibody CDI 315B raised against human breast carcinoma.

Our previous report revealed the production of monoclonal antibody (MoAb) CDI 315B by immunization of mice with tumor extract proteins of human invasive ductal breast carcinoma. In the present study we report on the immunocytochemical reactivity of this MoAb with formalin or methacarn fixed, paraffin embedded tissue sections and also with cell cultures. Among breast tissues, positive staining was detected in 88% (64 of 73) of primary breast carcinomas, 77% (7 of 9) of metastatic lymph nodes, 24% (8 of 33) of benign breast disease and 15% (2 of 13) of normal breast tissue. No immunostaining was detected with several other tumors, with the exception of melanoma, where 63% (5 of 8) of positive staining was found. On in vitro cell lines, positive reaction was detected only with breast carcinoma and melanoma cells, but not with other examined cell lines. On benign breast disease tissue sections, positive reaction was detected in areas with cell hyperplasia. On normal breast tissue sections MoAb 315B stained the epithelial cells of terminal ductuli. Since the MoAb 315B recognized some antigen present in the cytoplasm of most breast carcinoma cells, this MoAb may have potential application in diagnosis and management of breast cancer.

Immunohistochemical expression of monoclonal antibodies (epi-1 and myo-1) derived from human breast cancer against rat mammary tumors.

Immunohistochemical expression of monoclonal antibodies epi-1 and myo-1 derived from human breast cancer cell line (HBC-4W) was examined for DMBA-induced rat mammary tumors. Antibody epi-1 reacted with luminal epithelial cells while antibody myo-1 reacted with myoepithelial cells of the mammary glands in rats, respectively. The reactions with both antibodies were markedly visible, in particular, in the normal mammary gland, tumor-like lesions and benign epithelial mammary tumors in rats, which showed clear two-cell-type structures. Among malignant mammary tumors, adenocarcinoma was strongly positive with antibodies epi-1 and myo-1. However, squamous cell carcinoma and adenoacanthoma mainly reacted with antibody epi-1. On the other hand, the intercellular matrices of pleomorphic cell sarcoma and stromal areas of the normal mammary gland or epithelial tumors were positive with antibody myo-1.

Co-ordinate expression of the alpha-6 integrin laminin receptor sub-unit and laminin in breast cancer.

Interactions between cells and extracellular matrices are mediated in part by a family of heterodimeric molecules known as integrins. We have investigated, using immunohistology, the distribution of six integrin alpha sub-units in normal breast tissue and 26 breast carcinomas. Alpha-1 integrin (collagen/laminin receptor sub-unit) was detected in myoepithelium, but not in luminal epithelium nor in most (20/26) carcinomas. Its expression on fibroblasts was enhanced in desmoplastic stroma. Both benign and malignant epithelium showed uniform positive staining for alpha-2 (collagen receptor sub-unit) and for alpha-3 (collagen/fibronectin/laminin receptor sub-unit). All epithelium was negative for alpha-4 (sub-unit of a fibronectin receptor). Epithelial staining for alpha-5 (fibronectin receptor sub-unit) was weak in all samples. Alpha-6 (sub-unit of two integrin laminin receptors) showed conspicuous changes in all invasive carcinomas. In normal tissues, there was weak staining of epithelial cytoplasm with alpha-6 antibody and moderate cell membrane staining. Strongest staining was present in a basement membrane distribution. In carcinomas, loss of cytoplasmic and cell membrane staining was variable, but basal membrane staining was diminished or absent in all tumours. Loss of basal membrane staining for alpha-6 integrin corresponded closely to loss of immunoreactivity for its ligand laminin in invasive breast cancer.

Low expression of beta 1, alpha 2 and alpha 3 subunits of VLA integrins in malignant mammary tumours.

The Very Late Antigens (VLAs) are alpha beta heterodimeric transmembrane proteins mediating cell-substratum as well as cell-cell interactions. Changes in their expression and/or function seem to occur in a number of invasive carcinomas and may at least in part explain their abnormal patterns of growth and differentiation. Using monoclonal antibodies to the beta 1 (DH12, A1A-5), alpha 2 (B1.515) and alpha 3 (E1.56) chains, VLA-2 (alpha 2 beta 1) and VLA-3 (alpha 3 beta 1) were studied on cryostat sections of three fibroadenomas and 43 invasive breast carcinomas (29 ductal, 14 lobular) by the avidin-biotin complex immunoperoxidase technique. In non-neoplastic breast tissue and in fibroadenomas VLA-2 and VLA-3 were expressed by myoepithelial cells and on the basolateral surface of the luminal cells. There was weak or absent expression of alpha 2, alpha 3 and the common beta 1 chain in the majority of invasive carcinomas compared to the adjacent normal breast epithelium and preinvasive (in-situ) carcinomas. In addition, the expression of the alpha 2 chain of VLA-2 was reduced significantly (P less than 0.005) in the poorly differentiated ductal breast carcinomas (Grade III) compared to the well (Grade I) and moderately (Grade II) differentiated ductal tumours. These data give further evidence that loss or down-regulation of VLA-2 and VLA-3 occur relatively frequently in invasive cancers, and, at least in the invasive ductal breast carcinomas. Loss of an extracellular matrix receptor controlling growth and differentiation seems to be one of the abnormalities underlying the progression towards an undifferentiated morphology.

[Detection of antibodies to adipose tissue cell membranes in human blood]. / Obnaruzhenie antitel k kletochnym membranam zhirovoi tkani v syvorotke krovi liudei.

In serum of some healthy women and patients with fibroadenomatosis of the mammary gland antibodies to the cell membranes of adipocytes were detected. Interconnections between these antibodies and corresponding antigens in blood, on the one hand, and hormonal-metabolic status of probands, on the other hand, were observed. Possible autoimmune origin of phenomenon detected and its relation to the normal and pathological processes in adipose tissue are discussed.

Glial fibrillary acidic protein immunoreactivity in normal and diseased human breast.

Immunostaining for glial fibrillary acidic protein (GFAP) identifies a minor subpopulation of immunoreactive myoepithelial cells in the normal resting human breast. The GFAP-immunoreactive cells also express a panel of myoepithelial cell markers, including cytokeratin 14 (CK 14), vimentin, smooth-muscle-specific actin isoforms, nerve growth factor receptor (NGFR) and common acute lymphoblastic leukaemia antigen (CALLA). The percentage of GFAP-immunoreactive myoepithelial cells is greatly increased in various neoplastic and non-neoplastic diseases of the breast, being highest in adenomyoepitheliomas. Furthermore, in all the instances of fibroadenoma, phyllodes tumour, epitheliosis and gynaecomastia, a variable number of epithelial cells also acquires immunoreactivity for GFAP, vimentin, CK 14, NGFR and, to a lesser extent, for CALLA. Conversely, GFAP immunoreactivity has never been encountered in the malignant cells of the different types of breast carcinoma. These findings suggest that the expression of GFAP might be a (possibly transient) feature of proliferating epithelial and myoepithelial cells in breast diseases other than carcinomas.

Monoclonal antibody B72.3 in benign breast lesions.

It has been suggested that the monoclonal antibody B72.3 may be useful as a diagnostic tool in fine needle aspirates of breast masses because it recognises "tumour associated glycoprotein (TAG)-72". The antigen was sought in paraffin wax sections of 43 normal and benign breast biopsy specimens, using the avidin-biotin complex technique, to assess the extent of its presence in non-malignant tissue. Strong focal staining was seen in 21 (49%) cases. In 29 cases of fibrocystic change staining was present in 17 (59%). All areas of apocrine metaplasia were positive, as well as a few normal ducts and acini and occasional areas of adenosis. Focal positivity was present in five out of 12 foci of ductal epithelial hyperplasia and in three out of seven radial scars. Staining was absent in two areas of lobular hyperplasia, three areas of sclerosing adenosis, and in a focus of lactational change. Focal positivity was also seen in two out of five fibroadenomas and in two out of three intraduct papillomas. Five normal subareolar sections and a section of normal lactating breast were negative. It is concluded that B72.3 monoclonal antibody can show focal reactivity with a variety of normal and benign epithelial mammary structures, and it is doubtful that its use would be of any help in differentiating benign from malignant cells in fine needle aspirates.

Antigenic profile of mammary fibroadenoma and cystosarcoma phyllodes. A study using antibodies to estrogen- and progesterone receptors and to a panel of cell surface molecules.

Using serial frozen sections, monoclonal antibodies and an indirect immunoperoxidase method, 13 fibroadenomas (FA) and 3 cystosarcomas phyllodes (CSP) were analyzed for the expression of Egp34, HEA319-antigen, leucocyte differentiation antigens CD10, CD30, CD57, CD72, CDw75, and CD77, epidermal growth factor receptor (EGFR), estrogen (ER) and progesterone receptor (PR), and transferrin receptor (CD71). Egp34, CDw75, HEA319 antigen, CD10, and CD30 turned out to be consistently expressed in different cell types constituting FA and CSP and revealed that in malignant CSP the myoepithelial compartment acquires the ability to invade the stroma. Phenomenologically, the variable mode of expression of CD57 in myoepithelial cells, of CD77 in ductal epithelium, and of CD72 in both epithelial and stromal cells is suggestive for reflecting differences in their functional state but cannot be further interpreted at present. Expression of PR and ER was restricted to duct cells and was relatively independent, non-systematical. However, expression of ER and EGFR was inverse. This was also true for EGFR and CD71 in both duct cells and myoepithelial cells of FA. In contrast, stromal cells of FA were able to co-express EGFR and CD71 in the absence of PR and ER. This suggests a hormone-independent stimulation of the stromal cell compartment, possibly leading to local proliferation as the primary event in tumorigenesis of FA. In malignant CSP, however, the main proliferating cell is an abnormally mobile, HEA319 antigen-, CD10- and CD30-positive myoepithelial cell found to co-express ERFR and CD71 which is abnormal for this cell type but encountered in (myo-)fibroblasts of FA.

Breast carcinoma kinetics. Argyrophilic nucleolar organizer region counts correlate with Ki67 scores.

This study assessed the value of argyrophilic nucleolar organizer region (AgNOR) staining as a potential technique for the estimation of cell kinetics in conventional histology sections, in benign and malignant breast lesions. Using a silver staining technique and immunohistochemistry, the authors correlated the numbers of argyrophilic nucleolar organizer regions (AgNORs) and Ki67 scores in 70 breast carcinomas and 27 benign breast lesions. Epithelial cells in fibrocystic disease and fibroadenomas contained a mean of 2.65-6.8 small uniform AgNORs per cell, whereas malignant cells contained 4.6-26.9 frequently highly irregular AgNORs. In benign tissue, Ki67 scores ranged from 0 to 4%; in malignant tumors, Ki67 scores ranged from 3.0 to 98%. The correlation between AgNOR counts and Ki67 scores was highly significant (P less than 0.001). The authors concluded that AgNOR counts performed on routine formalin-fixed paraffin sections furnish significant kinetic information. Furthermore, the difference in AgNOR counts between benign and malignant tumors is such that they may be of diagnostic value.

Expression of HLA-A, -B, -C, -DR, -DP, -DQ, and of HLA-D-associated invariant chain (Ii) in non-neoplastic mammary epithelium, fibroadenoma, adenoma, and carcinoma of the breast.

Non-neoplastic mammary gland, 20 benign tumors and 206 carcinomas of the breast were immunohistochemically examined for expression of HLA-A, -B, -C, HLA-DR, -DP, and -DQ molecules and the HLA-D associated invariant chain (Ii). In contrast to cells from benign lesions, tumor cells of 51.2% of carcinomas had an abnormally low content of HLA-A, -B, and -C determinants ranging from reduction of antigenic density per cell (28.8%) over an incomplete (15.6%) to complete loss of antigens (6.8%). Associated with lymphohistiocytic stromal infiltrates, HLA-D/Ii determinants were found to be induced in benign duct and acinar epithelium after the order Ii greater than or equal to HLA-DR greater than or equal to HLA-DP greater than or equal to HLA-DQ. These antigens were also expressed, mostly noncoordinately, in 55.5% of carcinomas, and in 98 cases according to the above order. In 28.6%, Ii expression clearly exceeded HLA-D antigen expression; conversely, 6.2% contained HLA-DR+/Ii- tumor cell subsets. In breast carcinoma, the association of reduced HLA-A, -B, and -C expression and a noninduction of HLA-DR was highly significant (P less than 0.0009), suggesting an abnormal signal acting down-regulating on the expression of both classes of antigens. Because the modality of HLA-A, -B, and -C and HLA-D/Ii expression correlated with neither tumor type nor grade, it might be an independent parameter.

Tissue polypeptide antigen expression in human prostate tumors.

Thirty-seven specimens of benign and malignant prostatic tumors were studied for the localization of tissue polypeptide antigen (TPA) by an avidin-biotin-peroxidase complex technique. In addition, 23 metastases of prostatic carcinoma in other organs and 12 nonepithelial tumors of prostate also were studied. All benign and malignant tumors of epithelial origin, including their metastasis, stained positively. Nonepithelial tumors were uniformly negative. In the metastatic lesions, small foci of tumor cells and even single tumor cells could be identified by TPA staining. Immunohistochemical localization of TPA appeared to be a useful tool for assessing the micrometastases of prostatic carcinoma in other organs, especially lymph nodes, or elucidating the epithelial origin of an otherwise undifferentiated prostatic cancer.

[An immunohistologic study of reactive lymphocytes in breast tissue].

A series of T-cell specific monoclonal antibodies (Leu-4; CD-3, OKT-4; CD-4, OKT8; CD-8) and a B-cell specific monoclonal antibody (Leu-12; CD-19) were used to detect the localization and intensity of lymphocytes in breast tissue. Frozen sections of 12 fibrocystic diseases, 11 fibroadenomas and 17 carcinomas of the breast were examined during this study. CD-3 and CD-8 positive lymphocytes predominated in and around the ducts of the fibrocystic disease and the fibroadenoma. Similarly, almost all of the reactive lymphocytes around the cancerous tissue also were CD-3 and CD-8 positive suppressor/cytotoxic cells.

[The immunohistochemical localization of CA 15-3 in human breasts and their tumors].

The tissue distribution of CA 15-3, a breast carcinoma associated antigenic determinant, has been investigated by an avidin-biotin immunoperoxidase technique in the human breasts and in their tumors. In cases of scirrhous carcinoma, positive stainings for CA 15-3 were observed in almost all carcinoma cells. In cases of solid-tubular carcinomas and papillotubular carcinomas, the reaction was various and there was no correlation between the distribution of CA 15-3 and the clinical stage. Apical staining was recognized in both solid-tubular and papillotubular carcinomas, in benign breast tumors, and in normal breast tissues. CA 15-3 appears to be related to the degree of differentiation of the breast tissue.

Structural distinctions among human breast epithelial cells revealed by the monclonal antikeratin antibodies AE1 and AE3.

Two monoclonal antikeratin antibodies, AE1 and AE3, were used in indirect immunocytochemistry to examine keratin expression in normal, benign proliferative, and malignant human breast epithelium. Both antibodies reacted strongly with most luminal cells in ducts and acini of normal gland. While AE1 did not stain myoepithelium, AE3 recognized myoepithelial cells of ducts but not acini, implying a cytoskeletal difference between the myoepithelium of these two components. Moreover, the antibodies reacted differently with the myoepithelium of intracanalicular as compared with pericanalicular types of fibroadenomas. Tumour cells of infiltrating ductal carcinomas with a prominent intraductal component stained more homogeneously with AE1 and AE3 than those without intraductal growth. The results provide evidence for two phenotypes of myoepithelial cells and for the presence of cryptic keratin epitopes in human breast epithelial cells. The finding that neither AE1 nor AE3 is a universal detector of these cells has important clinical and experimental implications.