Prediction of prognosis in primary breast cancer by detection of a high molecular weight mucin-like antigen using monoclonal antibodies DF3, F36/22, and CU18: a Cancer and Leukemia Group B study.

Three monoclonal antibodies (MAbs) (DF3, F36/22, CU18) were used to monitor expression of distinct epitopes present within a family of mucin-like, breast carcinoma-associated molecules. Primary tumor specimens from more than 190 stage II breast cancer patients were evaluated for expression of the high molecular weight antigens. With a median follow-up of 6 years, patients whose tumors exhibited high immunoperoxidase staining scores (greater than 50% positive cells) with MAb DF3 had a superior disease-free survival ([DFS] 56% +/- 6% v 37% +/- 5% at 6 years; P = .0088) and overall survival ([OS] 72% +/- 5% v 59% +/- 5% at 6 years; P = .025). Staining scores with the other two antibodies did not correlate with improved prognosis. For MAbs DF3 and CU18, patients whose tumors exhibited predominantly apical cellular reactivity patterns had improved DFS, although differences reached conventional levels of statistical significance only with MAb CU18. In multivariate analyses, the prognostic value of MAb DF3 staining was independent of other identified prognostic factors. Furthermore, the concordance between primary and axillary lymph node metastases staining with each MAb was 73%, 80%, and 85% for MAbs DF3, F36/22, and CU18, respectively. These results suggest that staining with MAb DF3 identifies a group of node-positive women with a relatively favorable prognosis. Expression of the DF3 mucin-like glycoprotein is related to better differentiation, and staining with MAb DF3 provides an accurate and objective estimate of clinical outcome independent of histopathologic evaluation.

A new complication of AIDS: thoracic myelitis caused by herpes simplex virus.

Progressive thoracic myelopathy occurred in a patient with AIDS. Concurrent opportunistic infections included disseminated systemic cytomegalovirus, aspergillosis, and cutaneous herpes simplex virus (HSV). At autopsy, immune stains indicated that the myelopathy was caused by HSV type 2 infection of the spinal cord.

Immunohistochemical detection of a cross-reacting virus antigen in mouse mammary tumors and human breast carcinomas.

An indirect immunoperoxidase method was first used to localize mouse mammary tumor virus (MMTV) antigens in paraffin sections of mammary tumors of Paris RIII and CD8F1 mice. By using the same method, an antigen with cross-reactivity to a group-specific antigen (gp52, a 52,000 dalton glycoprotein) of MMTV was detected in paraffin sections of human breast carcinomas. The specificity of this reaction with antibody against MMTV was examined by absorption of the IgG with: a) purified gp52; b) several relevant and irrelevant viral preparations; c) normal human plasma, leukocytes, breast tissue, milk, actin, collagen, and hyaluronic acid; d) sheep erythrocytes, bovine mucin and fetal calf serum. Only MMTV and prufied gp52 eliminated the immunohistochemical reaction in human breast tumors. Positive reactions were seen in 73 of 191 (38%) breast carcinomas of various histopathologic types, while negative reactions were obtained in all 137 normal and benign cases tested. A positive reaction of uncertain specificity was observed in foci of apocrine metaplasia. With one exception, 99 carcinomas from 13 organs other than breast and eight cystosarcomas were negative.

Immunocytochemical localization of lysozyme and alpha-1-antichymotrypsin in the term human placenta: an attempt to characterize the Hofbauer cell.

The intracellular localization of lysozyme (LSZ) and alpha-1-antichymotrypsin (A1Ac), glycoproteins associated with macrophages, was used to confirm the monocytic lineage of the Hofbauer cell and to assess the maturity of its macrophage function. The peroxidase-labeled antigen method was used to localize these proteins, as well as immunoglobulins, light chains, and albumin, in Bouin-fixed, paraffin-embedded sections of 24 normal term placentas. The demonstration of the latter substances was used as an indication of passive diffusion or phagocytosis of serum proteins resulting in intracellular localization unrelated to synthesis. In all the placentas examined a strong cytoplasmic reaction for A1Ac was seen in the Hofbauer cells. The same cells on adjacent sections did not stain for LSZ, while the occasional maternal macrophage and numerous polymorphonuclear leukocytes in the intervillous spaces gave a positive reaction. The detection of A1Ac supports the contention that these cells are macrophages, previously suggested by their phagocytic capability and the demonstration of Fc receptors and nonspecific esterases. Since they do not appear to contain LSZ, a bactericidal enzyme, we propose that these cells are not fully differentiated macrophages, and the lack of this enzyme may have some relevance to the pathogenesis of certain placental infections.

Immunoperoxidase: a sensitive immunohistochemical technique as a "special stain" in the diagnostic pathology laboratory.

The present communication is a brief review of the origins, theory, and applications of a relatively new immunohistochemical technique that can be performed on routinely fixed, paraffin embedded tissues in which visualization by bright field light microscopy is feasible and which thus can be readily adapted to routine diagnostic work. The chief concern of this presentation is the practical diagnostic application of the immunoperoxidase technique, a method whose reputation as a sensitive investigative tool is well established.

Hepatitis B virus and myocarditis.

Myocarditis may be a serious extrahepatic complication of hepatitis. In this fatal case of serologically documented hepatitis B viral hepatitis, acute myocarditis was present, with histologic features consistent with a viral pathogenesis. Hepatitis B surface antigen was demonstrated by immunoperoxidase methods in small intramyocardial vessels, suggesting that hepatitis B virus infected the heart. The resulting inflammatory heart disease may have been caused either directly, by virus infecting the myocardium, or indirectly, by an immune-mediated mechanism.

Evidence of increased endothelial cell turnover in brain arteriovenous malformations.

OBJECTIVE: We hypothesized that human brain arteriovenous malformations (BAVMs) are nonstatic vascular lesions with active angiogenesis or vascular remodeling. To test this hypothesis, we assessed endothelial cell turnover in BAVMs. METHODS: We identified nonresting endothelial cells by use of immunohistochemistry for the Ki-67 antigen. From archived paraffin blocks, we selected BAVM vessels without intravascular thrombosis or embolic material in areas nonadjacent to the nidus edge. For controls, we used 50- to 100-microm diameter cortical vessels from temporal lobe cortex removed for epilepsy treatment. The Ki-67 index was calculated as a percentage of Ki-67-positive endothelial cells. The data were analyzed by the nonparametric Mann-Whitney test and reported as mean +/- standard deviation. RESULTS: Thirty-seven specimens that met the above criteria were selected. There were 26 +/- 15 vessels counted in each BAVM specimen versus 18 +/- 5 in each control cortex (n = 5). The mean Ki-67 index was higher for BAVM vessels than control cortical vessels (0.7 +/- 0.6 versus 0.1 +/- 0.2%; P = 0.005), which represented an approximately seven-fold increase in the number of nonresting endothelial cells. In the BAVM group, there was a trend for younger patients to have a wider variation and higher Ki-67 index than older patients; no trend was evident in the control group. CONCLUSION: Compared with control vessels, BAVM vessels have higher endothelial cell turnover, which suggests the presence of active angiogenesis or vascular remodeling in BAVMs.

Abnormal pattern of Tie-2 and vascular endothelial growth factor receptor expression in human cerebral arteriovenous malformations.

OBJECTIVE: Human cerebral arteriovenous malformations (AVMs) are speculated to result from abnormal angiogenesis. Vascular endothelial growth factor receptors (VEGF-Rs) and Tie-2 play critical roles in vasculogenesis and angiogenesis. We hypothesized that the abnormal vascular phenotype of AVMs may be associated with abnormal expression of VEGF-Rs and Tie-2. METHODS: We measured the expression of Tie-2, VEGF-R1, and VEGF-R2 in AVMs and normal brain tissue, using immunoblotting. To assess active vascular remodeling, we also measured endothelial nitric oxide synthase expression. CD31 expression was used to control for endothelial cell mass for Tie-2, VEGF-Rs, and endothelial nitric oxide synthase. Immunoblotting data were presented as relative expression, using normal brain tissue values as 100%. RESULTS: CD31 was expressed to similar degrees in AVMs and normal brain tissue (99+/-29% versus 100+/-20%, mean +/- standard error, P = 0.98). Tie-2 expression was markedly decreased in all AVMs, compared with normal brain tissue (16+/-9% versus 100+/-37%, P = 0.04). VEGF-R1 expression was decreased in four of five AVMs, but the difference between the mean values was not significant (35+/-8% versus 100+/-42%, P = 0.14). VEGF-R2 expression was decreased in all AVMs, compared with normal brain tissue (28+/-6% versus 100+/-29%, P = 0.03). There was no difference in endothelial nitric oxide synthase expression between AVMs and normal brain tissue (106+/-42% versus 100+/-25%, P = 0.91). CONCLUSION: AVM vessels exhibited abnormal expression of Tie-2 and VEGF-Rs, both of which may contribute to the pathogenesis of AVMs.

Cytomegalovirus retinitis: a manifestation of the acquired immune deficiency syndrome (AIDS).

Two homosexual males with the "gay bowel syndrome' experienced an acute unilateral loss of vision. Both patients had white intraretinal lesions, which became confluent. One of the cases had a depressed cell-mediated immunity; both patients ultimately died after a prolonged illness. In one patient cytomegalovirus was cultured from a vitreous biopsy. Autopsy revealed disseminated cytomegalovirus in both patients. Widespread retinal necrosis was evident, with typical nuclear and cytoplasmic inclusions of cytomegalovirus. Electron microscopy showed herpes virus, while immunoperoxidase techniques showed cytomegalovirus. The altered cell-mediated response present in homosexual patients may be responsible for the clinical syndromes of Kaposi's sarcoma and opportunistic infection by Pneumocystis carinii, herpes simplex, or cytomegalovirus.

Increased surface expression and shedding of tumor associated antigens by human breast carcinoma cells treated with recombinant human interferons or phorbol ester tumor promoters.

In the present study we have evaluated the effect of recombinant interferons, including leukocyte (IFN-alpha A), fibroblast (IFN-beta) and immune (IFN-gamma), and the tumor promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on the expression of tumor associated antigens (TAA) and class II HLA-DR antigens on human breast carcinoma cell lines. The effect of these agents on the shedding of a high molecular weight tumor associated glycoprotein, BCA-225, was also determined. All three interferons and TPA enhanced the expression of the Mr 180,000 carcinoembryonic antigen (CEA) and CEA-related TAA recognized by monoclonal antibody B1.1 in both T47D and MCF-7 human breast carcinoma cell lines. The three types of interferons and TPA differed in their absolute TAA-augmenting ability, even in single-cell subclones derived from MCF-7 cells and previously shown to display a differential susceptibility to IFN-alpha augmentation of B1.1 expression. In general, IFN-gamma was more effective than IFN-alpha, IFN-beta or TPA in augmenting the expression of TAA, CEA and BCA-225, and HLA-DR expression in T47D and MCF-7 cells. Differences were also apparent in the ability of the three interferon preparations and TPA to induce shedding of BCA-225 in T47D and MCF-7 cells and their subclones. As observed with TAA expression, IFN-gamma was the most effective preparation in inducing TAA shedding. IFN-gamma also induced the expression and the shedding of BCA-225 by a subclone of T47D cells, T47D cl 17, which normally displays a reduced expression of BCA-225 and does not spontaneously shed this TAA without exposure to IFN-gamma. Recombinant leukocyte interferon (IFN-alpha A) also enhanced BCA-225 expression on T47D cells grown as xenografts in nude mice in vivo. The results of the present study emphasize the complexity of potential antigenic responses which can be induced in human breast carcinoma cells when they are exposed to biological response modulators, including different types of interferon, and tumor promoting agents, such as TPA. This investigation also indicates that both classes of agents can differentially augment expression and/or shedding of TAA by specific breast carcinoma cell lines as well as subclones derived from the same breast carcinoma cell line.

Carcinoembryonic antigen: immunohistologic identification in invasive and intraepithelial carcinomas of the lung.

A total of 58 pulmonary lesions from 48 patients were examined for carcinoembryonic antigen (CEA). The three-layer immunoperoxidase procedure for antigen detection was used with a monospecific anti-CEA antiserum. The control serum was the same antiserum with its specificity removed by affinity chromatography. Normal goat serum was also used as a control. Carcinoembryonic antigen was present in the majority of pulmonary adenocarcinomas and generally absent in the squamous cancers. The major exception was in the well-differentiated squamous lesions where CEA was occasionally found in the keratinizing areas. Of special interest was the finding of CEA in all areas of intraepithelial squamous neoplasia studied.

Localization of IgE in tissues by an immunoperoxidase technique.

An indirect immunoperoxidase technique was used to study the content and distribution of IgE in formaldehyde solution-fixed, paraffin-embedded adenoid and nasal tissues of atopic and nonatopic persons. Adenoid tissue from 15 patients, and nasal tissue from five patients with symptoms of inhalant allergies and augmented serum levels of total and allergen-specific IgE were examined with this method. Adenoid tissues from five patients without allergic symptoms were studied for comparison. A rich content of IgE, largely within the cytoplasm of the plasma cells, was observed in tissues of atopic subjects. The sections from nonallergic persons contained few weakly staining IgE-positive cells. These observations provide anatomic support for the role of IgE in hypersensitivity reaction of immediate type. This immunoperoxidase technique, circumventing the principal shortcomings of the immunofluorescence procedures, affords a highly sensitive and practical approach to cellular and tissue localization of IgE.

Immunohistochemical evidence for RNA virus related components in human breast cancer.

The localization of antigenic components with cross-reactivity to a 52,000 dalton group specific glycoprotein (gp52) of the mouse mammary tumor virus (MMTV) in paraffin sections of human breast carcinomas is described using an indirect immunoperoxidase method. This method was first optimized on paraffin sections of mouse mammary tumors. The specificity of the reaction observed in the human tissues was established by absorption of the specific IgG with: a) purified gp52; b) several relevant and irrelevant viral preparations; c) normal human plasma, leukocytes, breast tissue, milk, actin, collagen, and hyaluronic acid; d) sheep erythrocytes, bovine mucin and fetal calf serum. Only MMTV and purified gp52 eliminated the immunohistochemical reaction in human breast tumors. Positive reactions were seen in 171 of 376 (45.5 percent) randomly selected breast carcinomas of various histopathologic types, while negative reactions were obtained in all 137 normal and benign cases tested. In those invasive tumors with an intraductal component, a higher percentage (63.9 percent) of positive cases was seen. A positive reaction of different specificity was observed in foci of apocrine metaplasia. With one exception, 99 carcinomas from 13 organs other than breast and eight cystosarcomas were negative.

Monoclonal antibodies: their importance to surgeons.

A tremendous technological advance occurred in 1975 when a method was developed to fuse two cells producing a "hybridoma" which secretes a single clone of antibody, having one immunoglobulin (Ig) class, one structure, one affinity, and one specificity for an antigenic determinant. Because monoclonal antibodies are more precise reagents than conventional antisera they open new doors to diagnosis and therapy of disease, and they are useful tools in research. The pathologist uses monoclonals in immunocytochemistry to determine tumor type; the surgeon uses monoclonals for immunosuppression in renal transplantation; the immunologist uses monoclonals to decipher cellular and humoral interactions that could not be appreciated with polyclonal reagents. This review outlines the background of monoclonal antibodies and some of their clinically important uses, both in vitro and in vivo. We also project into the future and describe chimeric antibodies and their possible uses.

Immunohistochemical characterization of fibrin(ogen)-related antigens in human tissues using monoclonal antibodies.

A new approach to study the distribution of fibrin(ogen)-related antigens was investigated using three different monoclonal antibodies (MAbs) and the avidin-biotin complex immunoperoxidase technique. MAb I8C6 recognizes B beta 1-42 peptide and can react with either fibrinogen or fibrin I; MAb T2G1 recognizes B beta 15-42 peptide and detects fibrin II but does not cross-react with fibrinogen; MAb GC4 reacts with Fragments D/DD derived from plasmin degradation of fibrinogen or fibrin but not with intact fibrinogen. The method can be applied to frozen or Bouin's fixed paraffin-embedded tissues obtained at biopsy, surgery, and autopsy. The distribution of the three antigens observed with the three MAbs was compared with that obtained with a polyclonal antiserum to fibrinogen and with the more conventional histochemical stains used in pathology to demonstrate fibrin deposits in tissues (Lendrum and PTAH). The staining observed with the three monoclonals clearly detected three different populations of fibrin(ogen)-related antigen in the tissues examined. The staining with MAb T2G1 specifically detected fibrin II with greater sensitivity than did conventional stains. The results of this study suggest that this method allows the molecular form of fibrin(ogen)-related deposits in tissues to be determined and this information may help to elucidate the role of fibrin in various disease states, such as atherosclerosis and renal disease, and in tumor growth and metastasis.

Patterns of immunocytochemically detected Z-DNA in the recrudescing testicular epithelium of the Turkish hamster (Mesocricetus brandti).

Z-DNA has been considered a labile but essential structural form of DNA in recombination and gene expression, two significant activities in mammalian seminiferous epithelium. The present study has utilized the recrudescing testes of Mesocricetus brandti to study in detail the potential Z-DNA sites in specific testicular cell types as detected by an immunoprobe. Testicular regression was physiologically induced by modifying environmental photoperiods and/or temperature. Partial atrophy of seminiferous epithelium occurred in all experimental groups but Sertoli cells persisted throughout regression. Recrudescence of testicular activity was marked in all experimental groups by characteristic sequences of reappearance of potential Z-DNA sites to a final positive or negative mature state of the cell type. It is suggested that Z-DNA is a functionally important from of DNA in many cell types of the active seminiferous epithelium of the Turkish hamster, and perhaps other mammals.

Identification and distribution of fibrinogen, fibrin, and fibrin(ogen) degradation products in atherosclerosis. Use of monoclonal antibodies.

Samples of normal and atherosclerotic vessels obtained from vascular and cardiothoracic surgery were examined for the distribution of fibrinogen/fibrin I, fibrin II, and fibrin(ogen) degradation products (Fragment D/DD) by using recently characterized monoclonal antibodies that recognize and distinguish the three molecular forms (MAbs 18C6, T2G1, and GC4, respectively) with the ABC-immunoperoxidase technique. In normal aortas, little fibrinogen/fibrin I or fibrin II was present and no fibrin(ogen) degradation products could be detected. In early lesions and in fibrous plaques, fibrinogen/fibrin I and fibrin II were distributed in long threads and surrounding vessel wall cells and macrophages. Fibrin(ogen) degradation products were not seen in early lesions. In fibrous and advanced plaques, fibrinogen/fibrin I, fibrin II, and fibrin(ogen) degradation products were detected in areas of loose connective tissue, in thrombus, and around cholesterol crystals. The results of this study suggest that increased fibrin formation and degradation may be associated with progression of atherosclerotic disease. The observed distribution of the different molecular forms of fibrinogen also suggests the possibility that the cells present in the lesions actively participate in the fibrinogen-to-fibrin transition within the vessel wall.