Detection in human breast carcinomas of an antigen immunologically related to a group-specific antigen of mouse mammary tumor virus (

Pulse-labeled RNA isolated from E. coli cells grown on limiting phosphate medium and phosphate-containing medium was analyzed by oligo(dT-cellulose chromatography and by Millipore binding assay for polyriboadenylate-containing RNA. Whereas poly(A)containing RNA amounted to as much as 15% of the total pulse-labeled RNA from cells grown on limiting phosphate medium, pulse-labeled RNA from cells grown on phosphate medium Extraction of RNA. The labeled cells were mixed with about 0.5 g of unlabeled cells grown under the same conditions, ground with 1 g of alumina, suspended in 2-3 ml of 0.05 M sodium acetate buffer containing 0.5 M NaCl, and clarified by centrifugation.The supernatant solution was mixed with 10% sodium dodecylsulfate to give a final concentration of 0.5%, treated with an equal volume of freshly distilled phenol, agitated vigorously for 30 sec, allowed to stand for 10 min, and agitated for 30 sec. The suspension was treated with 1 volume of CHC13, mixed vigorously for 30 sec, and allowed to stand for 5 min, and mixed for 30 sec. The aqueous layer obtained after centrifugation at 12,000 X g for 5 min was again deproteinized with CHC13, mixed with 2 volumes of 95% C2H5OH, and allowed to stand overnight at -20°. Precipitated RNA was collected by centrifugation, dissolved in 1 ml of 0.01 M Tris-Cl buffer (pH 7.5) containing 2 mM MgCl2, treated with electrophoretically pure DNase (10,g), incubated for 30 min at 40, and the solution was subjected twice to deproteinization with CHC13. RNA in the aqueous layer was precipitated with C2H5OH, collected by centrifugation at 12,000 X g, dissolved in 1 ml of Tris-Cl buffer (pH 7.5) containing 0.5 M KCl, and dialyzed twice against 100 volumes of the same buffer for a period of 1 hr. The yield of RNA was 30-40% based on radioactivity, and had a A260/280 ratio over 2.0.Oligo(dT)Cellulose Column Chromatography. The procedure described by Aviv and Leder (23)

We have shown [Mesa-Tejada, R., Keydar, I., Ramanarayanan, M., Ohno, T., Fenoglio (14). Because at most five sections are stained from each breast tumor, the percentage of positives detected must be an underestimate.It was of course gratifying to have extended to the protein level the relationship between human breast cancer and MMTV that was initially discovered in terms of nucleic acid sequence homology. The etiologic implications of these findings are of obvious interest but our immediate concern is the possibility that they might be used to generate clinically useful information. To this end, it was of interest to resolve certain issues regarding the nature of crossreactivity observed between the gp52 and the unique antigen found in the human breast cancers. In particular, it was of some importance to know whether the sugar or the protein moiety of the gp52 glycoprotein was responsible for its ability to block the immunological crossreaction with the antigen found in the breast cancer cells. Aside from its genetic implications, the outcome could materially influence the nature of our attempts to isolate and characterize the human tumorspecific antigen.It is the purpose of the present paper to present data that resolve this issue. The results establish that the antigenic crossreactivity between human breast cancer and gp52 glycoprotein resides in the polypeptide portion and not in the sugar residues of the latter protein. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. MATERIALS AND METHODS Enzymes

An indirect immunoperoxidase method is described, which can readily detect viral antigens in paraffin sections of primary, transplanted, and metastatic mammary tumors of mice. In addition to having the obvious advantage of not being limited to fresh specimens, immunoperoxidase staining of paraffin sections proved to be superior in many respects when compared with immunofluorescence and frozen sections. Immunoperoxidase staining of paraffin sections is permanent and provides the kind of histological detail required or recise cytological identification and localization with light microscopy. All of 25 tumors and 4 metastatic lesions showed evidence of glycoprotein gp52 as well as other mouse mammary tumor viral antigens. The pattern and intensity of the stain were related to the degree of histologic differentiation of the tumor. Wide variations in expression of viral antigens by individual malignant cells were observed within the same tumor.We have used the murine mammary tumor model to demonstrate the feasibility of employing viral proteins as systemic diagnostic indicators of the presence and status of a solid tumor. Mammary tumors in mice have been (1, 2) detected by increased plasma levels of gp52, a 52,000 molecular weight glycoprotein of the mouse mammary tumor virus (MMTV). It was subsequently shown (3) that surgical excision of the tumor is invariably followed within 9 days by a sharp (10-to 100-fold) decrease of the plasma gp52 levels. Further, the postsurgical behavior of the plasma gp52 is diagnostically and prognostically informative, as indicated by the following features: (a) All tumor regrowths were correctly diagnosed by increases in gp52 levels, and some were detected before they were found by palpation. (b) Relapses were accompanied by continued increases in plasma gp52 concentrations at rates that usually matched the speed of tumor development. (c) The only animals that remained tumor free at the termination of the experiment were those that maintained their gp52 levels at or below 15 ng/ml. (d) The probability of an individual's relapsing within a 2-week period is much higher if the gp52 level is initially above the mean and is almost certain if it is higher than two standard deviations above the mean.The evident usefulness of the plasma gp52 levels in the mouse model as both a diagnostic and monitoring device stimulated the attempt to achieve a similar situation in the corresponding human disease. Our earlier work revealed sufficient parallels between human breast cancer and the mouse model to warrant the effort involved. Thus, we established (4) that human breast cancers contain particles possessing many of the biochemical and biophysical features characteristic of the RNA tumor viruses. Further, we (5, 6) and others (7) have demonstrated the existence of a limited but clearly detectable homology between RNAs found in human breast carcinomas and the RNA genome of the mouse mammary tumor virus.The extension to the human disease of the diagnostic implications of the data obtained with t...

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.