Ultrastructural characteristics and variations in human mucinous breast carcinomas

Nineteen human mucinous breast carcinomas have been studied by electron microscopy in order to investigate the variations in the fine structure of this type of tumor. All the investigated tumors are characterized by a well-developed, rough endoplasmic reticulum and prominent Golgi complexes. The majority of tumor cells contain secretory granules. Approximately 50% (9/19) of the tumors have cytoplasmic dense core granules that are morphological identical to the neurosecretory granules found in APUD-cell derived tumors. Six out of the nine tumors react positively in a Grimelius staining for light microscopic argyrophilic granules. Two of the investigated breast carcinomas contain tonofilaments that are normally regarded as characteristic of squamous epithelium. It is concluded that mucinous breast carcinomas--that to this time have been regarded as a morphological homogenous group of tumors--are ultrastructurally characterized by heterogeneity.     

Rabbit leukocyte surface antigens defined by monoclonal antibodies

Several monoclonal antibodies (mAb) against rabbit leukocytes were characterized in binding and functional studies. mAb 1.24 stains thymocytes, bone marrow cells, peripheral T and B cells and blood monocytes. T cells express more 1.24 antigen than B cells. In the absence of added complement (C), mAb 1.24 inhibits alloantigen-, concanavalin A (Con A)-, and phytohemagglutinin (PHA)-, but not pokeweed mitogen (PWM)- or anti-immunoglobulin (Ig)-induced cell proliferation. It also strongly blocks anti-sheep erythrocyte plaque-forming cell responses. A second mAb, designated 4.B9, binds to 20% of thymocytes and to most, if not all, peripheral T cells and in vitro-activated T cell blasts. A third one, 10.B3, is reactive with the nearly entire thymocyte and a major peripheral T cell population. Two-color membrane immunofluorescence reveals the presence of a small population of peripheral blood leukocytes which bear surface Ig and are weakly stained by mAb 4.B9 and 10.B3. Without C, both 4.B9 and 10.B3 inhibit Con A- and PHA-induced mitogenesis, but have no effect on PWM-, antigen-, or alloantigen-induced cell proliferation. Depletion of 4.B9+ cells by panning or complement lysis completely abrogates proliferative responsiveness to antigen and alloantigen, significantly reduces responsiveness to the T cell mitogens Con A and PHA, but enhances that to the B cell mitogen anti-Ig. A fourth mAb, 12.C7, binds to 60% of thymocytes and to 10-30% of peripheral T lymphocytes at high-level fluorescence. T cell blasts obtained in mixed leukocyte reactions are partially stained by mAb 12.C7, while those obtained after Con A or PHA activation are not. In addition, mAb 12.C7 is completely unreactive with B cells or monocytes. Without complement, it does not seem to interfere with any of the in vitro functions tested. All antigens studied here do not appear to be expressed in nonleukon tissues, as they do not bind to erythrocytes and are absent from brain, heart, liver and kidney as shown by quantitative absorption analysis.     

Absence of microsatellite instability in mucinous carcinomas of the breast

Microsatellite instability (MSI) is a form of genetic instability that results from defects in DNA mismatch repair. MSI is reported to be rare in unselected breast cancers, however it is a common feature in subsets of colorectal, ovarian and endometrial cancers. In these anatomical sites, MSI-high carcinomas often display a mucinous histology. The aim of this study was to determine whether mucinous carcinomas of the breast would more frequently display MSI-high than invasive ductal carcinomas of no special type (IDC-NSTs). The expression of four MSI markers (i.e. MSH2, MSH6, MLH1 and PMS2) was immunohistochemically assessed in 35 mucinous breast carcinomas and 35 histological grade- and oestrogen receptor (ER) status-matched IDC-NSTs, and in a series of 245 invasive breast cancers. Cases were considered as potentially MSI-high if tumour cells lacked expression of at least two MSI markers and internal controls displayed nuclear staining. Nine mucinous carcinomas were microdissected and subjected to MSI analysis by PCR using the MSI markers BAT26 and BAT40. No immunohistochemical evidence of MSI-high was found in the 35 mucinous carcinomas and 35 grade- and ER-matched IDC-NSTs, and in the cohort of 245 invasive breast cancers. In addition, no evidence of MSI-high was observed by PCR analysis using the BAT26 and BAT40 markers in the nine mucinous carcinomas tested. Our results demonstrate that MSI-high phenotype is remarkably rare in invasive breast cancer, and that, in contrast to mucinous carcinomas of other anatomical sites, MSI is not a common event in mucinous carcinomas of the breast.     

Characterisation of renal antigens on distinct parts of the human nephron by monoclonal antibodies

Summary Ten monoclonal antibodies (TN 1-TN 10) directed against different renal antigens of distinct sites of the human nephron were derived from a fusion between P3-NS1/1-Ag4-1 mouse myeloma and spleen cells of a mouse hyperimmunized against isolated human kidney cells. Two of these reagents (TN 1, TN 10) were shown by immunoperoxidase labelling on frozen sections of five normal kidneys and of other selected human organs, as well as by immunofluorescence studies on normal peripheral blood cells and selected lymphohematopoietic cell lines, to detect antigens exclusively expressed on visceral glomerular or proximal tubular epithelial cells. The other eight antibodies were found to react with different determinants shared between renal structures, muscle cells, different epithelia, B-lymphocytes and granulocytes. In heterogeneous cultures of isolated kidney cells these monoclonal reagents could be used to identify distinct cell types of tubular origin. Thus such hybridoma-derived antibodies provide new tools to correlate structural characteristics of various renal epithelial cells with their functional properties and will contribute to the study of their influence on immunologically mediated kidney injuries in different forms of glomerulonephritis in man.This study was supported by two grants from the Deutsche Forschungsgemeinschaft, DFG, Mu 523/3-1 and SFB 120, Leukämieforschung und Immungenetik, Project A2 and A3     

Ki-67 expression in primary breast carcinomas and their axillary lymph node metastases: clinical implications

Proliferative activity of tumour cells assessed by immunohistochemical Ki-67 expression is one of several prognostic indicators in breast cancer. The major objective of this study was to investigate the prognostic impact of Ki-67 proliferative activity in the axillary lymph node metastases and in the matched primary breast carcinoma from 194 patients. There was a statistically significant up-regulation of Ki-67 protein in the metastatic deposit compared to where the primary tumour was found (p = 0.001). A low Ki-67 index in both the primary and the metastatic tumours was a favorable prognostic factor. A high index in both primary and metastatic lesion and an up-regulation from a low index in the primary tumour to a high index in the metastatic deposit represented an unfavorable prognostic factor. Multivariate analysis showed that Ki-67 expression in the metastases was a superior independent prognostic factor of clinical outcomes compared to that in the primary tumours. Ki-67 expression in ≥10% of carcinoma cells in the primary tumours and ≥15% in the nodal metastases seems to be optimal cut-off levels. Ki-67 is of value as an independent prognostic factor in breast cancer.     

T-Lymphocyte subpopulations in Crohn's disease: Definition by monoclonal antibodies

Summary Thirty-two patients with Crohn's disease (CD) and 32 age- and sex-matched controls were studied for T lymphocytes and T-lymphocyte subpopulations in the peripheral blood, using monoclonal antibodies defining the helper/inducer and the suppressor/cytotoxic compartment. T cells were reduced in patients with CD (P<0.05), and this reduction was more pronounced in patients with active discase (P<0.01). This T-cell deficiency involves helper and suppressor cells proportionally. The proportion of helper and suppressor cells in CD was independent of disease activity and therapy. We conclude that the T-cell deficiency in CD is a secondary event, and that there is no derangement in the immunoregulatory ratio (helper to suppressor cells) in CD which might have served as an explanation for a possible autoimmune mechanism in the pathogenesis of this disease.     

Rabbit monoclonal antibodies show higher sensitivity than mouse monoclonals for estrogen and progesterone receptor evaluation in breast cancer by immunohistochemistry

A novel generation of rabbit monoclonal antibodies for estrogen (ER) and progesterone (PR) receptor evaluation in breast cancer by immunohistochemistry has been released recently. We compared the novel RabMab anti-ER and anti-PR antibodies with the mouse monoclonal antibodies using a tissue microarray of breast carcinomas. Two cylinders (2mm diameter) of formalin-fixed, paraffin-embedded tissue were obtained from 24 invasive breast cancers and were immunostained using anti-ER mouse (1D5 and 6F11) and rabbit antibodies (SP1 and B644), and anti-PR mouse (PgR312 and PgR636) and rabbit antibodies (SP2 and B645). The immunohistochemistry was evaluated by considering positive those tumors in which more than 10% of the tumor cell nuclei stained independently on the staining intensity. Our results demonstrated that rabbit antibodies against ER have a similar staining pattern compared to the 6F11, but better than 1D5 from three different suppliers. The rabbit antibodies against PR (SP2 and B645) provide a stronger and sharper immunohistochemical signal compared to mouse antibodies (PgR636 and PgR312). Both ER and PR rabbit antibodies allow a lower cost per test because of higher working dilutions compared to mouse antibodies using the same procedure. The novel rabbit antibodies against ER and PR are highly sensitive for immunohistochemical testing of breast carcinomas.     

Increased heterogeneity of serum thyroglobulin in thyroid cancer patients as determined by monoclonal antibodies

Summary We investigated the immunological heterogeneity of plasma Tg in thyroid cancer patients using monoclonal antibodies in an immunoradiometric assay and a conventional RIA system with a polyclonal rabbit antibody. The results were compared with measurements of plasma Tg in patients with nonmalignant disease. We can demonstrate an increased immunological heterogeneity in tumor patients compared with patients with non-malignant thyroid diseases. In one case the Tg value measured by a monoclonal test system exceeded the value obtained by a polyclonal RIA system in the same sample by a factor of 25. It has to be further investigated whether this increase in heterogeneity is of diagnostic value in the follow-up of thyroid cancer patients.Abbreviations IRMA 11, IRMA 13 Immunoradiometric assay using mabTg 11 or mabTg 13 - mabTg monoclonal antithyroglobulin antibodies - Tg Thyroglobulin     

Cross-reactivity of mouse monoclonal antibodies produced by in vitro or in vivo immunization

The effect of different immunization schemes on the resulting antibody specificity was investigated. The cross-reactivity of monoclonal antibodies produced by in vitro vs. in vivo immunization was tested, using a solid phase enzyme immunoassay. Ten different monoclonal antibodies were tested against 15 different antigens. There was no difference in cross-reactivity between the two types of antibodies when tested against antigens coated onto the plastic wells in a buffer solution.When the antigens were dried onto the plastic wells the IgM monoclonal antibodies produced by in vitro immunization exhibited a somewhat different reactivity pattern. However, the assay design was shown to be of more importance than the immunization procedure when determining antibody specificities.     

Characterization of a peptide-loading compartment by monoclonal antibodies

Whether or not peptide-loading compartments are classical or specialized compartments of the endocytic pathway of antigen presenting cells is still a matter of debate. One way to solve this discrepancy would be to characterize specific markers for the peptide-loading compartment. We chose to generate monoclonal antibodies against the peptide-loading compartment that we previously characterized as lysozyme loading compartment (LLC) [Escola, J.M., Grivel, J.C., Chavrier, P., Gorvel, J.P., 1995. Different endocytic compartments are involved in the tight association of class II molecules with processed hen egg lysozyme and ribonuclease A in B cells. J. Cell Sci. 108, 2337; Escola, J.M., Deleuil, F., Stang, E., Boretto, J., Chavrier, P., Gorvel, J.P., 1996. Characterization of a lysozyme-major histocompatibility complex class II molecule-loading compartment as a specialized recycling endosome in murine B lymphocytes. J. Biol Chem. 271, 27360]. A preliminary screening by dot blot enabled us to identify several monoclonal antibodies recognizing the LLC and not early and late endosomes. One of these antibodies, the 20C4, was then characterized. It is directed against mature class II molecules of all murine haplotypes. By electron microscopy, 20C4 labeling was restricted to both the plasma membrane and the LLC. These reagents may be useful in the further characterization of the specialized function of these intracellular organelles.     

Development of AAV serotype-specific ELISAs using novel monoclonal antibodies

Adeno-associated viruses (AAV) have been developed and evaluated as recombinant vectors for gene therapy. More recently, due to the advantages they offer for gene transfer, several AAV serotypes have gained increasing interest. However, monoclonal antibodies for the characterization and quantitation of vectors derived from different serotypes are at present not available. Serotype-specific monoclonal antibodies (mAbs) against the capsids of the serotypes 1/6, 4 and 5 are described. These antibodies, designated as ADK1a and b, ADK4 or ADK5a and b, reacted specifically with the indicated serotype capsids in cell lysates. ADK 1a and b cross-reacted with its highly related AAV6 serotype, but not with the other serotypes tested. The new antibodies recognized exclusively assembled capsids and neither free nor denatured capsid proteins as shown by fractionation experiments. In immunofluorescence experiments, the mAbs stained only distinct intranuclear foci in cells expressing the capsid protein. The development of capture ELISAs for quantitation of AAV1 and 6, AAV4 or AAV5 capsids illustrates that these novel monoclonal antibodies provide valuable tools for characterization of vector stocks.     

Use of monoclonal antibodies in formol-paraffin sections

Summary Thirteen monoclonal antibodies (the VI-series) reactive with B- and T-lymphocytes, monocytes, granulocytes and erythrocytes were tested in sections fixed with formalin, formalin-sublimate or formalin-acetic acid. After fixation and embedding, most of the antigens were not detectable. However, VIE-G 4, a monoclonal antibody selective for glycophorin A, produced a strong reaction with erythroid cells in formalin fixed sections. The binding of the monoclonal antibodies VIM-D 5, VIM-2 and VIM-13 to myeloid, myelomonocytic and monocytic cells became either more intense or was inhibited by preincubation of the sections with pronase, trypsin, papain or neuraminidase. Following enzyme digestion, the monocyte-specific antibody VIM-13 reacted selectively with some of the germinal centre cells.     

The use of colloidal gold for screening monoclonal antibodies to cell surface antigens

An immunogold method in Terasaki plates is described which allows accurate and sensitive visualization of the binding of monoclonal antibodies to cell surface antigens and is suitable for large scale screening. Monolayers of fixed cells are prepared in the wells. The binding of monoclonal antibodies is detected by a protein A gold complex. The cell-bound gold can be visualized by either optical or transmission electron microscopy. The results obtained with various monoclonal antibodies are presented.     

Characterization of monoclonal antibodies against human lactoferrin

The iron-binding glycoprotein human lactoferrin (hLF) is involved in the host defense against infection and is a modulator of inflammatory reactions. We generated monoclonal antibodies (mAbs) to hLF as tools to assist both structure-function studies and the development of recombinant human lactoferrin for applications in human health care. Binding experiments with ten distinct anti-hLF mAbs to tryptic and recombinant hLF fragments in ELISA and/or on immunoblots revealed that five mAbs bound to conformational epitopes residing in the N-lobe (residues 1 to 334), whereas the other five bound to C-lobe conformational epitopes (residues 335 to 692). None of the mAbs bound to hLF denatured upon reduction. Monoclonal antibody E11 appeared to bind to the arginine-rich N-terminus of hLF, which is the binding site for heparin, bacterial lipopolysaccharide, human lysozyme, DNA and receptors. The dissociation constant of the distinct mAbs for hLF ranged from 0.5 to 18 nM, without differences in affinity for unsaturated or iron-saturated hLF, indicating that the conformational changes subject to incorporation of iron do not seem to affect the exposure and/or conformation of the antibody epitopes. The mAbs did not bind to human transferrin, a protein closely related to hLF in size, primary amino acid sequence and structure. Two C-lobe specific mAbs, E2 and E8, cross-reacted with bovine and/or porcine lactoferrin, indicating that human, bovine and porcine lactoferrin share antigenic determinants. This panel of mAbs will be used to develop quantitative and qualitative immunoassays for hLF and to delineate which regions of hLF are relevant to its anti-infective and anti-inflammatory properties.     

Genomic imbalances detected by comparative genomic hybridization are prognostic markers in invasive ductal breast carcinomas

Genomic imbalances detected by comparative genomic hybridization are prognostic markers in invasive ductal breast carcinomas
Aims : The aim of this work is the study of the prognostic significance of the chromosomal aberrations described in a series of invasive ductal breast carcinomas.
Methods and results : We analysed by comparative genomic hybridization a group of 70 formalin-fixed paraffin-embedded invasive ductal breast carcinomas. Aberrations showed a frequency similar to previous studies using frozen tumours. Interestingly, we identified gains involving 6q16-q24 more frequently than in other series. We analysed the association among the chromosomal imbalances, 11 histopathological factors, relapse rate and overall survival of patients. Associations showed 16q losses as a potential marker of good prognosis, as they were more frequent in node-negative ( P =0.025) and in oestrogen-positive tumours ( P  < 0.001). Furthermore, 100% of bcl-2+ tumours presented this aberration compared with 29.3% in bcl-2– ( P =0.014). 1q, 11q, 17q and 20q gains were associated with poor prognosis: 95% of cases with 1q gains were bigger than 20 mm ( P =0.041). Tumours with 1q and 11q gains showed a higher relapse rate ( P =0.063; P =0.066). Within the good prognosis group of lymph node-negative patients, 17q and 20q gains identify a subgroup with increased relapse rate ( P =0.039).
Conclusions : Chromosomal imbalances, together with histopathological factors, may help to predict outcome in breast cancer patients.     

Plasmodium falciparum: characterization of defined antigens by monoclonal antibodies.

Monoclonal antibodies directed against Plasmodium falciparum detect stage-specific, species-specific and common antigenic determinants of Plasmodia. These antibodies provide new tools for purification and characterization of Plasmodium falciparum antigens in relation to future procedures for immunoprophylaxis.     

Expression of the antigen detected by the monoclonal antibody Ca 19.9 in human breast tissues

Summary The incidence and significance of the expression of the antigen defined by the monoclonal antibody Ca 19.9 (Sialyl Le^a) has been assessed in human breast tissue. Frozen and formalin-fixed, paraffin embedded specimens of normal, hyperplastic, pregnant breast and carcinomas were examined using an immunoperoxidase technique.Ductal and acinar epithelium of normal and hyperplastic tissues showed variable reactivity in frozen sections but there was a reduction in staining in comparable samples after fixation and processing, such that in many instances only focal ductal epithelium reacted. A distinctive feature in the pregnant breast was the absence of staining in acini showing differentiated secretory activity, despite a reaction in adjacent non-secretory acini and ducts.The overall incidence of detection of the Ca 19.9 antigen in breast carcinomas was 62%, but in half of these only a small number of cells stained. A significant relationship between expression of Sialyl Le^a and poor differentiation of carcinomas was identified, but there was no correlation with local lymph node status. In contrast to the non-malignant tissue fixation and processing had little effect on the reactivity of carcinomas. It is suggested that this difference may be quantitative in nature, with malignant breast showing much greater expression, or be related to organisation of the antigen.The observations concerning carcinomas and pregnant breast indicate that the synthesis of the Ca 19.9 antigen is related to the state of differentiation and functional activity of human breast.     

Correlation between active E-rosettes and antigens defined on peripheral lymphocytes by OKT 4 and OKT 8 monoclonal antibodies

A relationship has been sought between the classical marker, active E-rosette and the surface antigens of T-lymphocytes, defined by OKT 4 and 8 monoclonal antigens, in the peripheral blood of normal human people. It was found that no significant correlation existed in the case of OKT 4 antigen; conversely the OKT 8-positive cells were significantly enriched after E-rosette-forming cell isolation (about 2.5 times in comparison with the negatively selected cells). In this latter case, some partial correlation could exist between SRBC-high affinity of T-lymphocytes and the subsets defined by OKT 8 antigen.