Benign Mammary Tumors Research Articles

Type I insulin-like growth factor receptor function in breast cancer.

Experimental evidence suggests an important role of the type I IGF receptor (IGF-IR) in breast cancer development. Breast tumors and breast cancer cell lines express the IGF-IR. IGF-IR levels are higher in cancer cells than in normal breast tissue or in benign mammary tumors. The ligands of the IGF-IR are potent mitogens promoting monolayer and anchorage-independent growth of breast cancer cells. Interference with IGF-IR activation, expression, or signaling inhibits growth and induces apoptosis in breast cancer cells. In addition, recent studies established the involvement of the IGF-IR in the regulation of breast cancer cell motility and adhesion. We have demonstrated that in MCF-7 cells, overexpression of the IGF-IR promotes E-cadherin-dependent cell aggregation, which is associated with enhanced cell proliferation and prolonged survival in three-dimensional culture. The expression or function of the IGF-IR in breast cancer cells is modulated by different humoral factors, such as estrogen, progesterone, IGF-II, and interleukin-1. The IGF-IR and the estrogen receptor (ER) are usually co-expressed and the two signaling systems are engaged in a complex functional cross-talk controlling cell proliferation. Despite the convincing experimental evidence, the role of the IGF-IR in breast cancer etiology, especially in metastatic progression, is still not clear. The view emerging from cellular and animal studies is that abnormally high levels of IGF-IRs may contribute to the increase of tumor mass and/or aid tumor recurrence, by promoting proliferation, cell survival, and cell-cell interactions. However, in breast cancer, except for the well established correlation with ER status, the associations of the IGF-IR with other prognostic parameters are still insufficiently documented.

Effect of wheat bran fiber on the development of mammary tumors in female intact and ovariectomized rats treated with 7,12-dimethylbenz(a)anthracene and in mice with spontaneously developing mammary tumors.

We examined the effect of consumption of graded increases of dietary fiber (soft white wheat bran) on the development of mammary gland carcinomas in intact female Sprague-Dawley rats during the promotion stage of carcinogenesis, induced with 7,12-dimethylbenz(a)anthracene (DMBA). The percent of rats with mammary carcinomas, the total number of mammary carcinomas and the mean number of mammary carcinomas per rat were reduced significantly at all fiber levels examined compared to rats fed a control diet. Inclusion of 9.6% fiber in the diets of ovariectomized rats that had been treated with a single i.v. dose of 2.5 mg DMBA/100 g body weight 2 weeks prior to removal of the ovaries resulted in a significant decrease of carcinomatous and benign mammary tumors compared to ovariectomized rats fed a control diet. Development of spontaneous mammary carcinomas in virgin C3H/HeOuJ female mice and growth of a transplantable mammary gland tumor in such mice were reduced by inclusion of 9.6% fiber in the diet, a reduction that was significant or just barely missed significance, depending on the source of the fiber. Our observations provide evidence that inclusion of soft white wheat bran in the diet is effective in the suppression of mammary gland tumorigenesis in an array of experimental animal models.

Immunohistochemical distribution of type IV collagenase in normal, dysplastic and neoplastic canine mammary gland

The distribution of type IV collagenase (cIVase) was evaluated immunohistochemically in a series of normal, dysplastic and neoplastic canine mammary glands, as well as in lymph node metastases. In normal and dysplastic mammary tissues, and benign mammary tumours, cIVase was observed in myoepithelial cells, but in malignant tumours it was mainly localized in the cytoplasm of carcinoma cells and fibroblasts close to infiltrating neoplastic cells. No significant difference was observed between carcinomas of different histological sub-types. These results suggest that the distribution of cIVase is a potentially useful indicator of malignancy in canine mammary tumours.

Transcriptional Down-regulation of the Metastasis-inducing S100A4 (p9Ka) in Benign but Not in Malignant Rat Mammary Epithelial Cells by GC-factor

The S100-related calcium-binding protein S100A4 (p9Ka) is expressed at a low level in rat mammary epithelial cells from normal mammary gland and benign mammary tumors. In transgenic mice, expressed rat S100A4 transgenes co-operate with the activated c-erbB-2 oncogene, neu, to form metastatic mammary tumors. Elevated levels of S100A4 (p9Ka) in cultured benign rat or mouse mammary epithelial cells are associated with the induction of metastatic capability. A cis-acting sequence related to the consensus recognition sequence of GC-factor, 1,300 base pairs upstream of the start site of transcription of the rat S100A4 gene, acts as a cis-acting inhibitor of transcription of the S100A4 (p9Ka) gene in a low S100A4 (p9Ka)-expressing benign rat mammary epithelial cell line, but not in highly expressing rat mammary epithelial cell lines. There is an inverse relationship between the level of S100A4 (p9Ka) mRNA and the level of GC-factor mRNA in a range of rat mammary cell lines. The results suggest a novel mechanism for regulating the expression of the mRNA encoding an S100 protein.

Number of Nucleoli and Nucleolar Organizer Regions per Nucleus and Nucleolus — Prognostic Value in Canine Mammary Tumors

Twenty-eight canine mammary tumors were evaluated for histopathologic classification as recommended by the World Health Organization and silver-binding nucleolar organizer region (AgNOR) and nucleolus counts. Samples of surgically excised tumors and tumors taken at necropsy were fixed in neutral formalin, embedded in paraffin, and cut into 1-3-microns-thick sections. Two sections were taken from each tumor: one was stained with hematoxylin and the other was treated with the silver staining technique for the demonstration of AgNORs. After histopathologic classification, the number of nucleoli and the number of AgNORs/nucleus and AgNORs/nucleolus were determined. Statistical analysis (Student's t-test) showed a significant difference in the mean number of nucleoli (P < 0.005), mean number of AgNORs/nucleolus (P < 0.001), and mean number of AgNORs/nucleus (P < 0.005) between benign and malignant canine mammary tumors. There was no significant differences between metastatic and nonmetastatic malignant tumors.

The identification of metastasis-related gene products in a rodent mammary tumour model.

Breast cancer is currently the most common form of serious malignancy in women, affecting approximately 1 in 9 of the female population in the Western World [l]. The etiological background of breast cancer is currently largely unclm, however, diet, reproductive lifestyle and heredity are thought to be important risk factors [2]. The most life-heatening aspect of many forms of malignancies, including breast cancer, is the ability of cells from the primary tumour to disseminate to distant sites of the body. This dissemination, or metastasis as it is known, is a complex multistep process requiring a number of genetic interactions and a variety of gene products [3]. In order to study the process of metastasis at the molecular level, a rodent mammary tumour model has been developed in our laboratory. This completely syngeneic system centres around the use of the highly-inbred Furth-Wistar rat [4]. A cell line, designated Rama 37 was isolated from a benign mammary tumour that developed following Uearment of the Furth-Wistar rat with dimethylbenz[a]antene (DMBA). This cell line, when injected into the mammary fat pads of the syngeneic host produces benign, nonmetastatic, encapsulated tumours. A metastatic derivative of the Rama 37 cell line has previously been produced by transfecting this cell line with genomic DNA fragments from a metastasizing human breast carcinoma derived cell line [5]. The resultant transfected cells, when injected into the mammary fat pads of the Furth-Wistar rat, developed metastases at a high incidence. From one such lung metastases, a cell line was isolated designated Ca2-5-LT1 which when introduced into the host also metastasized [5]. In an aftempt to identify those gene products that are associated with the progression from the benign tumour producing cell line, Rama 37 to the metastatic derivative, Ca2-5LTl a variation of the subtractive hybridisation technique has been used [6-71. This technique was performed in a manner that permitted the identification of those mRNAs expressed more highly in the Ca2-5-LT1 cell line when compared to the Rama 37 cell line. Following the implementation of the subtractive hybridisation procedure and the production of a subuacted cDNA library, Northern blot analysis was performed using individual subtracted cDNAs as hybridisation probes. From these studies a number of subtracted cDNAs were identified which corresponded to mRNAs that were expressed more highly in the metastatic cell line when compared to the non-metastatic counterpart. The nucleic acid sequence of these cDNA molecules was then determined and compared to the cDNA sequences stored on the Genbank data base (Table 1). . . Table 1 Iden t l f r ca t lonc ted cDNAs c -

New insights in the molecular mechanism of progestin-induced proliferation of mammary epithelium: Induction of the local biosynthesis of growth hormone (GH) in the mammary gland of dogs, cats and humans

In contrast to the protective, anti-proliferative, action of progestins on the development of endometrium cancer, progestins may have local stimulatory and inhibitory effects on the proliferation of mammary epithelium. Until now there was no final molecular explanation of this discrepancy. Prolonged treatment of dogs with depot medroxyprogesterone acetate (DPMA) or with proligestone (PROL) results in enhanced plasma concentrations of growth hormone (GH), insulin-like growth factor (IGF)-I, IGF-II and IGF-binding proteins, together with the development of benign mammary tumours. The stimulated plasma GH levels do not have the typical pulsatile secretion pattern, and are not sensitive to stimulation with GHRH or to inhibition with somatostatin. The autonomous secretion can be inhibited by the anti-progestin RUU-486. The source of progestin-induced plasma GH levels has been demonstrated to be the canine mammary gland where progestins induce the expression of the gene encoding GH. The expression of the GH gene is restricted to focal areas of hyperplastic epithelium as shown by immunohistochemistry, and is predominantly located in single positive epithelial cells with an intermediate position between luminal- and myo-epithelium. Progestin-induced fibroadenomatous changes in the mammary gland of cats are also associated with locally enhanced GH expression. In both normal, benign and malignant mammary tumours of humans GH mRNA expression has been demonstrated by RT-PCR. The presence of GH mRNA is associated with the presence of immunoreactive GH as shown by immunohistochemistry. Sequence analysis revealed 100% homology to the pituitary expressed GH gene. In malignant mammary tumours of humans and dogs GH expression is also found in specimens negative for progesterone receptors as measured by ligand binding. It is concluded that the gene encoding GH is expressed in the mammary gland of a variety of species, including man. This appears to represent a contribution to the molecular explanation of the action of progestins on proliferation of mammary epithelium. It needs, however, to be proven whether this local biosynthesis of GH in the mammary gland is the cause of the local stimulatory effect of progestins on the proliferation of mammary epithelium.

Transplantation of a cell line derived from a canine benign mixed mammary tumour into nude mice.

The MCM-B2 canine mammary cell line was serially transplanted into nude mice. The tumour masses consisted of elongated pleomorphic cells of varying size in the first to third passages; oval cells, becoming rounder, in the sixth to eighth passages; and cord-like, glandular and duct-like structures with compact radiating projections in the ninth and tenth passages. Ultrastructural and immunohistochemical examination of round cells confirmed their epithelial cell nature, but the morphology of the elongated and oval cells was identical with that of the original cell line. The findings suggest that the MCM-B2 cell line is a multipotential stem cell or is derived from glandular differentiation of mammary gland.

Detection of tumor-associated antigens in sera of canine cancer patients by monoclonal antibodies generated against canine mammary carcinoma cells

Two murine monoclonal antibodies (MAbs), 1A10 and SB2, generated against a canine mammary carcinoma cell line, were used in a competitive enzyme-linked immunosorbent assay (ELISA) to measure tumor-associated antigens (TAAs) in canine serum samples. Sera were tested from disease-free dogs and from dogs diagnosed with mammary carcinoma, non-mammary carcinoma, sarcoma, benign mammary tumor, benign non-mammary tumor, or non-neoplastic disease. Serum antigen concentrations measured by ELISA were expressed as inhibitory units (IU). The upper limit of normal, defined as the mean plus 2 SD of the TAA concentration in disease-free dogs, was 20 IU with antibody 1A10 and 22 IU with antibody SB2. Compared with disease-free dogs, the frequency of TAA-positive sera was significantly greater ( P < 0.05) among dogs with mammary or non-mammary carcinoma when tested with MAbs 1A10 or SB2, and also with sarcoma when tested with MAb SB2. Testing a serum sample with both antibodies rather than just one increased the sensitivity of the competitive ELISA for TAA detection. The presence of TAA in serum might serve as a useful marker for certain types of carcinomas or sarcomas in canine cancer patients.

Krankheitsbewältigung/Lebensqualität und immunologische Parameter bei Patientinnen mit Mammakarzinom und benignen Tumoren

Maladaptive coping of the cancer illness leads to considerable psychosocial burden which can-as a chronic stress factor-impair various immune functions, e.g. cellular immunity. In a collective of 118 patients suffering from mammary carcinoma and 48 patients suffering from benign mammary tumors Coping was measured with the EORTC-MAC-scale and Quality of life with three questionnaires. Out of the immunologic variables the lymphocyte subpopulations were determined with flow cytometry. Immunglobulines, neopterin, C-reactive protein, and herpes serology were determined using standard methods. At follow up a slight increase of the mean vales of sum-scores is observed for the adverse coping mechanisms, like helplessness/despair and fear. On the other hand, the values for the coping styles fatalism and denial decrease. Significant correlations are seen between anxious attitude and number of natural killer cells (CD16 and CD56). It must be pointed out that area of social contact show an inverse correlation in patients with mammary cancers: a strong improvement correlates with diminished natural killer cells as well as reduced activated killer cells. The aim of this study is to determine whether a high risk group can be defined using these parameters and if these parameter can be influenced by psychotherapeutic interventions such as establishing "Coping-support-groups".

More about Mammary Tumors in Beagles Given Radium-226

A recently published paper from this laboratory (1) reported an excess of malignant mammary tumors among female beagles injected with 226Ra as compared with female control beagles (among 226Ra dogs, there were 34 expected and 66 observed, P < 0.005). A corresponding excess was not seen for benign mammary tumors. There seemed to be no difference between dogs given 226Ra and controls in the appearance time for the first malignant mammary tumor, but there was an indication that the timing of subsequent mammary cancers (three or more per animal) was affected by 226Ra exposure. Neither ovariectomy nor exposure to diagnostic X rays could be shown to be associated with the difference between controls and dogs given 226Ra with regard to mammary malignancies. It was proposed in that report (1) that the increased risk of mammary cancer might have been a result of (a) initial deposition of 226Ra in sensitive tissue, or (b) transient deposition of 226Ra progeny in the mammae, or (c) a general effect on the immune system of skeletal a-particle irradiation by radium and its progeny that might reduce the resistance to expression of mammary neoplasia. Since our report was accepted for publication, we have had the opportunity to examine the mammary tumor experience among comparable female beagles given injections of either 241Am or 239pu. Both of these radionuclides are a-particle emitters and also irradiate the skeleton. However, no such differences in mammary tumor frequency between dogs given 241Am or 239pu and controls could be detected. These data have some relevance to the third of the three possibilities given above that were discussed by Bruenger et al. (1). Reports dealing with the analysis of soft-tissue tumors in our beagles given 241Am or 239Pu have been submitted for publication or are in preparation. Not included in the published version of our paper on mammary tumors in dogs given 226Ra was an analysis of the comparison between irradiated beagles and controls with respect to the number of separate dates on which mammary tumors were diagnosed in the individual animals. An excess of malignant tumors in the dogs given 226Ra could have been a result of (a) a greater average number of dates on which tumors were diagnosed, (b) a larger average number of tumors diagnosed at each diagnosis date or (c) a combination of both (a) and (b). Table I exhibits the results of this comparison. It appears from the data in Table I that there cannot be established a difference in the average number of dates on which tumors were diagnosed between controls and irradiated animals: Therefore, it seems more probable that the observed difference in the number of malignant tumors reported by Bruenger et al. (1) may have been a result of a larger average number of tumors diagnosed in 226Ra dogs on any one day. For many years, it has been the policy of our group to use the word dose to denote the energy imparted to matter and to designate a corresponding value that has the units of gray or rad, exclusively. To avoid confusion, we have tried (not always successfully) to use the word dosage to denote such other concepts as the amount of radioactivity adminis-

Immunohistochemical localisation of cytokeratin and vimentin intermediate filament proteins in canine mammary tumours

Monoclonal antibodies to human stratifying keratin (StK), simple keratin ( sk), broad spectrum keratin ( bsk) and vimentin were applied to 47 canine mammary tumours (three benign hyperplasia, 26 benign mammary tumours, one malignant mixed tumour, two malignant complex tumours, seven lobular carcinomas, three papillary carcinomas and five squamous cell carcinomas). In benign hyperplasia the SK antibody reacted with acinar and duct epithelial cells; the StK and bsk antibodies reacted strongly with duct epithelial cells and myoepithelium. Neoplastic epithelial cells in the canine mammary tumours expressed mainly keratins of stratified epithelia rather than those of simple epithelia. Myoepithelial cells in complex and mixed tumours can express StK and vimentin. The finding might be helpful in the assessment of complex tumours where stromal reactions may be confused with myoepithelial neoplastic proliferation. Stromal mesenchymal tissues, including precartilage in complex tumours, and clearly differentiated myoepithelial cells always stained positively for vimentin. Occasional carcinoma cells stained positively for vimentin.

Immunohistochemistry with keratin, vimentin, desmin, and alpha-smooth muscle actin monoclonal antibodies in canine mammary gland: benign mammary tumours and duct ectasias.

Duct ectasias (n = 2) and different types of benign canine mammary tumours (n = 19) were studied immunohistochemically with monoclonal antibodies (MoAbs) directed against various human keratin types (K), alpha-smooth muscle actin, vimentin, and desmin. In the duct ectasias and in most tumours the epithelial structures revealed an inner and outer cell layer. The inner cell layer was characterized by labelling with K 7, 8, 18, 19 and mostly also with K 4 and/or K 10 MoAbs. The outer cell layer was almost invariably labelled by K 14, K 14 and 17, and a-smooth muscle actin MoAbs. The labelling patterns of both duct ectasias and tumours corresponded largely to the patterns observed in normal mammary gland tissue, although a more distinct heterogeneity was seen. Tumours histomorphologically assumed to be of a myoepithelial origin did not show immunohistochemical features of myoepithelial cells. The myoepithelial nature of the vast majority of spindle-shaped cells present in the adenomas of the complex type and in the fibroadenomas of the benign mixed type could not be confirmed immunohistochemically. These cells, however, unequivocally expressed vimentin, suggesting proliferation of stromal cells in these tumours, which in the fibroadenomas of the benign mixed type may show metaplasia to bone or cartilage. In the duct ectasias and in some tumours, a fraction of elongated stromal cells, probably representing myofibroblasts, was labelled with the alpha-smooth muscle actin MoAb.

Induction of the metastatic phenotype in rat mammary benign tumour cells by the gene for p9Ka a novel calcium-binding protein

Induction of the metastatic phenotype in rat mammary benign tumour cells by the gene for p9Ka a novel calcium-binding protein

Epidermal growth factor in human breast cancer, endometrial carcinoma and lung cancer. Its relationship to epidermal growth factor receptor, estradiol receptor and tumor TNM

Epidermal growth factor (EGF) and its receptor (EGFR) were measured in 60 breast cancers (BC), 6 benign mammary tumors (BM), 8 samples of normal breast (NB), 6 endometrial carcinomas (EC) and 30 lung cancers (LC). EGF was measured in plasma, saliva and urine from 20 patients with BC, before and after tumor excision, and in 8 patients with metastatic disease. The median EGF in BM and BC was significantly higher ( P < 0.05) than in NB. No significant correlation between EGF and EGFR was found in BC. Neither tumor excision nor the spreading of the disease significantly modified the EGF concentrations in biological fluids. In LC there was an inverse relationship between EGF and EGFR ( rs = −0.36; P = 0.09), which disappeared in normal lung. It is concluded that EGF may play a role in malignant transformation; however, the weak correlation between EGF and EGFR lessens the importance of EGF in either autocrine or paracrine stimulation of tumor growth.

Effects of high‐fat diet on incidence of spontaneous tumors in Wistar rats

In a 2.5-year carcinogenicity study, two groups, both including male and female Wistar rats, were fed two different diets with 4% and 16% fat. In addition to 4% soybean oil, the high-fat diet contained 12% mono- and diglycerides, of which 85% was stearic acid and 13% palmitic acid. There was no difference in food consumption, body weight, weight gain, and longevity between the two groups. A statistically significant increase in the incidence of tumors in the high-fat group was seen in fibroadenoma of the mammae (female, p = 0.05). No statistically significant difference was seen when the incidence of benign mammary tumors (adenomas and fibroadenomas) was combined, just as the overall incidence of mammary tumors (adenomas, fibroadenomas, and adenocarcinomas) was not significantly different between the groups. A statistically significant decrease in the incidence of tumors in the high-fat group was seen in adenoma of the parathyroid gland (male, p = 0.04) and medullary carcinoma of the adrenal gland (male, p = 0.04). Combining the incidence of benign and malignant tumors of the adrenal medulla led to a further increase in the level of significance (p = 0.02). The present study showed that a high-fat diet influenced the tumor incidence in certain organs of rats. However, the overall differences in tumor incidence between rats fed the low- and the high-fat diet are considered marginal. Therefore we were not able to confirm or deny the hypothesis that a high-fat diet promotes the development of cancer. It should be noted that, in our study, fat accounted for about 30% of the total energy in the high-fat diet. This is much below the amount of fat normally found in the western diet but corresponds well to the level recommended for human intake. In addition, the rats fed the high-fat diet did not gain more weight, even though no difference was recorded in food consumption (g/kg body wt) between the groups.

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.

Caffeine inhibits development of benign mammary gland tumors in carcinogen-treated female Sprague-Dawley rats

The purpose of this study was to assess the influence of caffeine on the incidence of benign mammary tumors in carcinogen (DMBA) treated female Sprague-Dawley rats. Four different animal models were used in these studies, i.e., the administration of DMBA to: [1] 55 day old virgin rats; [2] 53 day old ovariectomized, estrogen treated virgin rats; [3] 135 day old virgin rats and [4] 135 day old parous rats. A high incidence of benign mammary fibroadenomas was observed in each of the four animal models. In addition, in the estrogen treated ovariectomized animals, a high incidence of secretory mammary gland cysts was observed. Caffeine (500 mg/L drinking water) was administered daily throughout the study commencing 3-31 days after carcinogen treatment. Caffeine treatment significantly (P less than 0.05 to P less than 0.001) reduced the incidence of benign mammary fibroadenomas in the 55 day old virgin rat model (P less than 0.01), in the 53 day old estrogen treated ovariectomized virgin rat model (P less than 0.05 to P less than 0.001) and in the 135 day old virgin rat model (P less than 0.05). The number of benign mammary fibroadenomas was reduced by caffeine in the 135 day old parous rat model but this reduction was not significant (P less than 0.10). In addition, in the estrogen treated ovariectomized virgin rat model, caffeine significantly (P less than 0.05 to P less than 0.001) reduced the incidence of mammary gland cysts. Caffeine treatment either increased or had no significant effect on body weight gains, depending upon the animal model.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometric DNA measurement and cytomorphometric analysis of formalin fixed rat mammary tumours

Archival paraffin embedded material was used to examine whether additional quantitative criteria would be helpful to discriminate between histologically benign and malignant rat mammary tumours. To this end nuclear DNA content expressed as DNA ploidy index (DI) was measured using flow cytometry (FCM). A total of 63 benign and malignant mammary tumours were investigated. Thirteen out of 38 (34%) mammary carcinomas were DNA aneuploid against 0 out of 25 benign mammary tumours. Aneuploidy was not significantly increased in tumours showing histological signs of greater malignancy such as cribriform-comedo type or invasive growth. In addition to DI other quantitative criteria indicative for malignancy, such as mitotic count and nuclear morphometric characteristics, were estimated in 24 benign and malignant tubulopapillary tumours, a category where the histological classification may be difficult. It appeared that five out of nine noninvasive tubulopapillary carcinomas and six out of seven invasive carcinomas had abnormal values for either DI, mitotic count or nuclear area or for a combination of these parameters. Each single parameter however was abnormal only in a minority of the malignant tumours. In this respect our data are in accordance with the fact that rat mammary carcinomas are clinically and histologically less malignant than their human counterparts.

Conserved antigen expression in epithelial tumors recognized by monoclonal antibody 4A9 generated against canine mammary carcinoma cells.

Hybridoma-derived murine monoclonal antibodies (MoAbs) were generated by fusing P3X63-Ag8.653 myeloma cells with splenic cells from BALB/c mouse which had been immunized with viable canine mammary adenocarcinoma cells, CMT-2. Fifteen MoAbs were shown to react with immunizing cells in indirect immunofluorescence (IFA) and enzyme-linked immunosorbent (ELISA) assays. The reactivity of one IgM MoAb, designated 4A9, was evaluated. The antigen recognized by 4A9 on CMT-2 cells appeared to be localized both in cell membrane and cytoplasm against fixed and unfixed preparations by IFA. The 4A9 MoAb was found to bind with four of five canine mammary carcinoma cell lines while no binding was detected with normal fibroblastic cell lines. In vivo tissue distribution of 4A9 antigen was evaluated by indirect immunoperoxidase (IP) assay against formalin-fixed, paraffin-embedded sections of normal and neoplastic tissues. 4A9 MoAb reacted strongly to moderately with 75% of mammary carcinomas, moderately to weakly with 57% of benign mammary tumors, and strongly with squamous cell and perianal gland carcinomas (100%), interstitial cell tumors (100%), transitional cell carcinomas (43%), lung adenocarcinomas (40%), colon carcinomas (33%), and pancreatic adenocarcinomas (20%). Moderate to weak staining was detected with granulosa cell tumors (25%) and apocrine gland adenocarcinomas (50%). Strong reactivity with perianal gland carcinomas contrasted to no reactivity with perianal gland adenomas. No immunostaining was detected with a large variety and number of normal adult and fetal tissues tested; negligible and very restricted staining was observed in a few adult and fetal tissues. Normal mammary gland was negative. Since the antigen is expressed on the cell surface and in the cytoplasm of most mammary carcinoma cells and a variety of other epithelial tumor cells, the 4A9 antibody may have potential application in diagnosis and management of canine mammary cancer and a variety of other epithelial tumors.