Avidin-biotin Complex Immunoperoxidase Method Research Articles

Myointimoma of the penis

Myointimoma is an uncommon, benign soft-tissue tumor derived from the intimal cells of blood vessels. Since little is known about this rare tumor entity, our aim is to describe an additional case and to perform the first literature review on this topic. A 49-year-old Caucasian man presented with a 12-month history of a palpable, firm, solitary lesion involving the glans penis. On physical examination, there was a 1 cm palpable, endophytic well-circumscribed nodule located to the left side of glans penis, close to the coronal sulcus, with disease-free external urethral orifice. The patient was submitted to complete excisional biopsy. A skin rhombus measuring 1.1 × 0.8 × 0.3 cm was removed and the biopsy sample, fixed in 10% formaldehyde, sent to Pathology. At the 18-month follow-up visit, the patient was clinically disease free. Histopathology revealed a multinodular intravascular proliferation of the corpus spongiosum. This myointimal proliferation comprised bland predominantly spindle cells in an abundant fibromyxoid stroma. Immunostains for smooth muscle actin (1A4), cytokeratins (AE1/AE3, CAM5.2), and CD34 were carried out using the avidin-biotin complex (ABC) immunoperoxidase method. Lesional cells displayed positivity for smooth muscle actin and negativity for cytokeratins and CD34. Myointimoma is confirmed to be a penile benign lesion that may be adequately treated with excisional biopsy. Even after incomplete or marginal removal, the penile lesion has been shown to remain stable overtime or regress. Differential diagnosis is essential to exclude similar histologic entities that could be more aggressive or have possible systemic implications.

Interaction between Esophageal Squamous Cell Carcinoma and Adipose Tissue in Vitro

Esophageal squamous cell carcinoma (ESCC) develops within the squamous epithelial layer and invades the submucosa to the subadventitia that has adipose tissue (AT). AT seems critical to ESCC progression, but the underlying mechanism is unknown. We aimed to address the association between ESCC and AT invitro. ESCC cells were cultured on rat or human subcutaneous AT-embedded or -non-embedded collagen gel. AT promoted the growth of ESCC cells and inhibited their apoptosis. AT promoted the expression of the squamous differentiation marker involucrin in ESCC cells. AT accelerated the expression of invasion-related factors in poorly differentiated ESCC cells only. AT promoted the expression of phosphorylated-insulin-like growth factor-1 receptor in ESCC cells, whereas it inhibited that of the human epidermal growth factor receptor 2. Insulin-like growth factor-1, but not leptin, adiponectin, or resistin, promoted and inhibited the growth and apoptosis of ESCC cells, respectively. In turn, ESCC cells decreased the production of these adipokines in AT and the number of preadipocytes and mesenchymal stem cell-like cells, which developed from AT. These results suggest that i) AT may influence the progression of ESCC with increased growth or invasion and decreased apoptosis through insulin-like growth factor-1/insulin-like growth factor-1 receptor signaling, ii) AT may affect human epidermal growth factor receptor 2-targeted therapy; and iii) the cancer cells may affect adipokine production in AT.

A clinicopathological study of diagnostically challenging meningioma mimics

The occurrence of neoplastic and nonneoplastic dural based masses that mimic meningiomas is infrequent and may not be considered during radiological and intraoperative analysis. We describe single institute study of 20 such rare cases. This study included total of 20 cases of meningioma mimics. The clinical, radiological and histopathological findings were evaluated. Tissue fixed in 10% formalin was routinely processed and 5 μ thick sections were cut and stained with hematoxylin & eosin. Histochemistry and immunohistochemistry using avidin-biotin complex immunoperoxidase method was done wherever indicated. In the present study group, 15 were male and 5 female with a male: female ratio of 3:1. The age ranged from 14 to 78 years. Radiologically all these lesions were extra-axial in location, predominantly hypointense on T2W, isointense on T1W images and showed intense homogenous enhancement on contrast administration. Four cases were in pediatric age group with histopathological diagnosis of Rosai Dorfman disease, medulloblastoma, hemangiopericytoma and malignant melanoma. In the adult population, the histopathological diagnoses were hemangiopericytoma, undifferentiated sarcoma, extraskeletal osteosarcoma, Rosai Dorfman disease, medulloblastoma, and metastases from systemic malignancies. Of the total 6 cases of metastases 1 was nonseminomatous germ cell tumor from a primary in testis, 1 was adenocarcinoma from an unknown primary, and 4 were adenocarcinoma from lung. There was a single case of dural based frontal lobe malignant melanoma with congenital hairy nevi on anterior chest wall, scalp, anterior abdominal wall and inguinal region. As the management and biologic behaviour of many of the MM are different, it is essential to familiarize ourselves to them.

Tissue Tropism of African Horsesickness Virus in the Chicken Embryo Demonstrated With the Avidin–Biotin Complex Immunoperoxidase Method

In horses, African horsesickness virus (AHSV) exhibits marked tropism for certain microvascular endothelia and components of the mononuclear phagocyte system. In this study, the tropism of a field isolate of AHSV serotype 5 was studied in 24 chicken embryos. Histopathology on embryonic tissues harvested with 12 hour intervals revealed progressive changes associated with endothelial damage. Immunolabeling demonstrated viral antigens in the microvascular endothelium of the spleen, lungs, and the mesenchymal connective tissue at the base of the neck, from 24 hours post inoculation. Subsequently, specific immunolabeling increased steadily in endothelia of these and other tissues such as skeletal and cardiac muscle, gastrointestinal smooth muscle, mesonephric glomeruli, liver, subcutis and feathers. Positive immunolabeling was also occasionally observed in circulating mononuclear cells and in Kupffer cells in the liver. It was concluded, that this isolate of AHSV displayed similar tissue tropism in the chicken embryo as in the horse.

T cell receptor heterodimer (alpha beta/gamma delta) expressions in diffuse panbronchiolitis

Several reports have indicated that T cells bearing gamma delta T cell receptor (TCR) are major effector cells in mucosal immunity of the murine intestinal tract. In humans, abnormal distribution of gamma delta + T cells has also been found in inflammatory bowel diseases. We studied TCR heterodimer expressions in lymphocytes infiltrating the peripheral airways in patients with diffuse panbronchiolitis (DPB), because DPB is histologically characterized by chronic inflammation in bronchioles and could be a model for investigation of the significance of gamma delta + T cells in persistent inflammation of the human respiratory tract. Open lung biopsy specimens were obtained from 7 patients and peripheral blood lymphocytes (PBL) from 9 patients. Immunohistochemically, we detected alpha beta and gamma delta TCR by avidin-biotin complex immunoperoxidase method, utilizing BMA 031 and TCR delta 1 monoclonal antibody, respectively, PBL were analyzed by flow cytometry. Histological analysis revealed that DPB lesions mainly involved the bronchiolar walls with marked infiltration of small round cells. Many T cells were distributed in the submucosal area, although few T cells infiltrated the epithelium. Most of the T cells in the bronchioles expressed alpha beta TCR (98.7%), whereas only 1.3% expressed gamma delta TCR. There was no significant difference in the percentage of gamma delta + T cells in PBL between DPB and age- and sex-matched controls (6.7% vs. 9.1%). These results suggest that the increase of T cells bearing alpha beta TCR played a major role. However, the possibility that the decrease of T cells bearing gamma delta TCR caused reduced mucosal defense against external antigens, leading to persistent inflammation of the lower respiratory tracts in DPB, is not excluded.

Prognostic impact of Deoxyribonucleic acid (DNA) image analysis cytometry and immunohistochemical expression of Ki67 in surgically resected non-small cell lung cancers

Background: The aim of the present study was to evaluate the prognostic significance of DNA ploidy and Ki67 expression in non-small cell lung carcinoma (NSCLC). Methods: This prospective study included 96 patients with stages I–IIIA NSCLC who underwent surgical excision. DNA image analysis cytometry was applied on imprints. Calculation of the DNA index (DI) and the 5c exceeding rate (5cER) was performed and the histograms were classified as peridiploid, peritetraploid, and x-ploid–multiploid. The Ki67 immunoreactivity was determined according to the avidin–biotin complex immunoperoxidase method. Results: DNA histogram classification disclosed 30 peridiploid cases, 15 peritetraploid and 51 x-ploid–multiploid. Forty-eight cases (50%) had 5cER > 5%. The Ki67 immunoreactivity was below 25% in 53 tumors (62.4%) and above 25% in 32 (32.6%). Our results revealed the existence of a statistically significant relationship of DNA ploidy with nodal status ( p = 0.042) and grade ( p = 0.005). Adenocarcinomas and large cell carcinomas were more frequently encountered in x-ploid–multiploid tumors as compared to squamous cell carcinomas, which were more frequently peridiploid ( p = 0.003). 5cER showed statistically significant association with nodal status ( p = 0.037). Univariate analysis with respect to survival revealed significant association with stage ( p < 0.001), nodal status ( p < 0.001), tumor status ( p < 0.001), DNA ploidy ( p = 0.008) and 5cER ( p = 0.0124). Multivariate analysis revealed stage and ploidy status as independent factors: peridiploid tumors were associated with better survival as compared to x-ploid–multiploid tumors ( p = 0.022). Conclusion: Our results suggest that DNA ploidy, as determined by image analysis, provides an independent prognostic parameter for patients with NSCLC and thus, could be used to identify a subset of patients with more aggressive tumors.

Application of an immunodiagnostic method for improving preoperative diagnosis of nodular thyroid lesions

Thyroid cancer is the most common endocrine malignant disease, but preoperative diagnosis remains a challenge. Fine-needle aspiration cytology has greatly improved the clinical management of thyroid nodules, but the preoperative characterisation of follicular lesions is very difficult. Many patients are thus referred to surgery more for diagnosis than for therapeutic necessity. We undertook an international multicentre study to assess the usefulness of immunohistocytochemical staining for two potential markers of malignant thyrocytes. Expression of galectin-3 and CD44v6 was tested on 1009 thyroid lesions (tissue specimens and cytological cell-blocks) and 226 fresh cytological samples obtained preoperatively by ultrasound-guided fine-needle aspiration of thyroid nodules (prospective analysis). The test used monoclonal antibodies specific for CD44v6 and galectin-3, the indirect avidin-biotin complex immunoperoxidase method, and 3-amino-9-ethyl-carbazole as substrate. The sensitivity, specificity, positive predictive value, and diagnostic accuracy of this test method (for coexpression of the two markers) in the prospective analysis were 88%, 98%, 91%, and 97%, respectively. The sensitivity and specificity of galectin-3 immunodetection alone in discriminating benign from malignant thyroid lesions were more than 99% and 98% respectively, and the positive predictive value and diagnostic accuracy were 92% and 99%. The integration of galectin-3 immunostaining with conventional cytomorphological and clinical diagnostic procedures represents a sensitive and reliable diagnostic approach for preoperative identification of thyroid carcinomas. This test method improves the diagnostic accuracy of conventional cytology and provides the molecular basis for a new nosological assignation of the not yet classified thyroid neoplasms of indeterminate malignant behaviour.

Transforming growth factor beta receptor induction in herniated intervertebral disc tissue: an immunohistochemical study.

Transforming growth factor beta (TGF-beta) is a potent inducer of angiogenesis and fibrogenesis. There is presently little information about the pathophysiological function of TGF-beta in herniated disc tissue. In order to analyze the cellular role and activation of TGF-beta after disc herniation we immunostained frozen material from 38 disc herniation operations and from eight macroscopically normal discs from organ donors. Polyclonal TGF-beta-I, TGF-beta-II and TGF-beta receptor type II antibodies were used with the avidin biotin complex (ABC-) immunoperoxidase method. All the herniated discs were TGF-beta immunopositive. Such immunoreactivity was mainly associated with disc cells. In a few samples, capillaries were also TGF-beta immunopositive. Immunopositivity was similarly observed in the control discs. To analyze possible differences between the two groups, we calculated the ratio of immunopositive disc cells. For all three antibodies, a statistically significantly (Mann-Whitney test, P = 0.0001) higher number of disc cells showed immunopositivity in the herniated discs. The increase in TGF-beta receptor immunopositivity suggested induction of TGF-beta receptors in herniated discs. Our results support an active regulatory role for TGF-beta in disc cell metabolism.

PS2 Expression in infiltrating ductal carcinoma of the breast correlates with oestrogen receptor positivity but not with histological grade and lymph node status

This investigation was carried out to gain insight into the prevalence of pS2 expression in invasive ductal breast carcinoma in the Malaysian population and its correlation with oestrogen receptor (ER) protein expression and tumour aggressiveness. Seventy consecutive infiltrating ductal breast carcinomas treated with mastectomy and axillary lymph node clearance were investigated, using the standard avidin-biotin complex immunoperoxidase method with microwave antigen retrieval and commercial monoclonal antibodies (Dako), for expression of pS2 and human ER. This was correlated against histological grade (modified Bloom and Richardson) and the presence of axillary lymph node metastasis of these carcinomas. Four (5.7%) were grade 1, 40 (57.1%) grade 2 and 26 (37.1%) grade 3 tumours. A total of 45 (64%) showed histological evidence of axillary lymph node metastasis. Forty (57%) were ER-positive, while 31 (44%) were pS2-positive. There was a statistically significant correlation between pS2 and ER expressions (chi2-test with Yates correction: P<0.005). There was no correlation between pS2 expression and histological grade (P>0.1) and the presence of lymph node metastasis (P>0.1). Our findings support the views that pS2 may be a co-marker of endocrine responsiveness in invasive breast cancer and that it does not influence breast cancer biology in terms of potential for metastatic spread.

The myoepithelial immunophenotype in 135 benign and malignant salivary gland tumors other than pleomorphic adenoma.

We have previously studied the immunoreactivity of 3 novel smooth muscle-specific proteins, alpha-smooth muscle actin, smooth muscle myosin heavy chains, and calponin, to assess myoepithelial differentiation in pleomorphic adenomas. To further expand our knowledge of myoepithelial differentiation in other benign and malignant salivary gland tumors. Formalin-fixed paraffin sections of 135 salivary gland tumors with associated normal glands were stained with monoclonal antibodies using the avidin-biotin complex immunoperoxidase method and enzymatic and microwave heat-induced epitope retrieval. In adenoid cystic carcinomas and epithelial-myoepithelial carcinomas, all 3 markers exclusively highlighted the myoepithelial cell components and the epithelial cells were entirely negative. No immunostaining was detected in canalicular adenomas, oncocytomas, Warthin tumors, acinic cell carcinomas, mucoepidermoid carcinomas, squamous cell carcinomas, and polymorphous low-grade adenocarcinomas. Salivary duct carcinomas and adenocarcinomas, not otherwise specified had a distinctive pattern of uniform periductal staining of reactive myofibroblastic cells, and in salivary duct carcinomas some ducts retained a peripheral immunoreactive myoepithelial cell layer. Immunoreactivity for these 3 smooth muscle-specific proteins confirms the known neoplastic myoepithelial component of adenoid cystic carcinomas and epithelial-myoepithelial carcinomas. The consistently positive staining pattern in adenoid cystic carcinomas may be diagnostically useful in discriminating histologically similar but consistently negative polymorphous low-grade adenocarcinomas. Periductal linear staining in adenocarcinoma, not otherwise specified and salivary duct carcinomas is distinctive and appears to represent a tight cuff of myofibroblasts associated with the infiltrating glands.

A modified immunoperoxidase assay for detection of antibody porcine reproductive and respiratory syndrome (PRRS) virus in swine sera.

MARC-145 cell monolayers infected with PRRS virus were fixed in 3% neutral buffered formalin and embedded in paraffin. The sections were stained by avidin-biotin complex immunoperoxidase (ABC) method. Test sera were applied to sections as primary antibodies. The positive reactions were detected by ABC method and indirect immunofluorescent assay (IFA). There was good correlation between ABC and IFA, and the titers in ABC were higher than those in IFA. The present results indicate that the immunohistochemical staining is a useful test for the detection and quantitation of PRRS virus antibody in swine sera as well as IFA.

Detection of antibodies against Actinobacillus pleuropneumoniae serotypes 1, 2, 5 and 7 using the immunohistochemical staining.

Whole cells of Actinobacillus pleuropneumoniae (A. pleuropneumoniae) serotype 1, 2, 5 or 7 attached to fibrins were fixed in 10% neutral buffered formalin and embedded in paraffin. The sections on a slide glass were stained by the avidin-biotin complex immunoperoxidase (ABC) method. Test sera were applied to sections as primary antibodies. The serum antibodies against A.pleuropneumoniae (serotypes 1, 2, 5 and 7) were measured by the ABC method and complement fixation (CF) test. There was good correlation between the ABC and CF tests. The present results indicate that the immunohistochemical staining is as useful as the CF test for the detection and quantification of antibody in swine sera.

Ontogeny of endogenous secretion of immunoreactive-thyrotropin releasing hormone by the human placenta.

We studied endogenous production of immunologically active TRH (ir-TRH) by human placenta throughout gestation. Fragments (20 g) of placentae obtained between 7 and 41 weeks' gestation were incubated in TC-199 media with or without TRH degrading enzyme inhibitors (1 mM of dithiothreitol, 200 microm O-phenanthroline, 0.8 mM EDTA) at 37 C for 24 h. TRH was quantitated by RIA. Release of ir-TRH was also studied in an in vitro model of dually-perfused isolated lobule of human term placenta. Cellular localization of TRH was performed by staining early-, mid- and late-gestation placentae with anti-TRH rabbit polyclonal antibody, using the indirect avidin-biotin complex immunoperoxidase method. TRH was produced by placental fragments from 7 to 41 weeks' gestation. Placental TRH secretion was maximal between 7-12 weeks gestation both in presence (655 +/- 79 pg/10 mg protein) and absence (423 +/- 75 pg/10 mg protein) of enzyme inhibitors. Secretion of TRH declined with increasing gestation both with (y = 779 - 15x; r = 0.90; P < 0.001; n = 15) and without (y = 525 - 12x; r = 0.87; P < 0.001; n = 15) enzyme inhibitors. In the perfusion experiments, endogenous TRH was released predominantly into the fetal circulation, and its concentration was markedly higher in the presence of enzyme inhibitors (146 +/- 27 vs. 34 +/- 7 pg/mL; P < 0.001). Immunostaining of chorionic villi localized TRH to the cytoplasm of the syncytial layer, and the intensity declined with advancing gestational age. These data suggest that immunoactive TRH is produced by human placenta begin at 7 weeks gestation, and production declines with gestational age.

Detection of the Apoptosis-suppressing Oncoprotein bcl-2 in Salivary Gland Lymphoma

The bcl-2 proto-oncogene encodes an inner mitochondrial membrane protein that blocks apoptosis and programmed cell death in human lymphoid tissue. In this study a monospecific anti-human bcl-2 antibody that is reactive in formalin-fixed tissues was used with an avidin-biotin complex immunoperoxidase method to evaluate 41 cases of lymphoproliferative disorders of the salivary gland. The study cases were 26 primary salivary gland lymphomas (including 21 B-cell lymphomas four T-cell lymphomas and one true histiocytic lymphoma) and 15 cases of myoepithelial sialadenitis. Bcl-2 expression is restricted to the mantle zone and interfollicular lymphocytes around reactive germinal centers of myoepithelial sialadenitis. Seventeen of the 21 B-cell lymphomas were positive for bcl-2, and were composed of mucosa-associated lymphoid tissue (MALT), centrocytic, centroblastic-centrocytic and centroblastic lymphomas. Noticeably, all 11 cases of MALT lymphoma were bcl-2 positive. In contrast, staining for bcl-2 was present in only one of four cases of T-cell lymphomas and was negative in one true histiocytic lymphoma. The expression of bcl-2 protein was also investigated in the ductal systems and epimyoepithelial islands of salivary glands from patients with malignant lymphoma and myoepithelial sialadenitis. While salivary ducts in eight of 15 cases of myoepithelial sialadenitis immunostained for bcl-2, epimyoepithelial islands showed bcl-2 expression in only five cases of myoepithelial sialadenitis. We found that ductal cells in the salivary gland from patients with primary non-Hodgkin's lymphomas expressed bcl-2 protein. It was of interest that epimyoepithelial islands in all cases of MALT lymphoma displayed bcl-2 expression whereas other subtypes of B-cell lymphoma, T-cell lymphoma and true histiocytic lymphoma were invariably negative. These results indicate that bcl-2 is expressed in a wide variety of non-Hodgkin's lymphomas, especially when all 11 cases of MALT lymphoma are bcl-2 positive. Epimyoepithelial islands in MALT lymphoma express this oncoprotein, and their ability to induce bcl-2 synthesis resulted in the prevention of apoptosis and prolonged cell survival. Furthermore, the expression of bcl-2 protein in the lymphoma cells may be responsible for the induction of bcl-2 expression in the adjacent epimyoepithelial islands through a lymphocyte chemical mediator.

Apoptosis is inhibited early in the dysplasia-carcinoma sequence of Barrett esophagus.

To evaluate the alteration of apoptosis in the esophageal epithelium during the esophagitis-Barrett esophagus-adenocarcinoma sequence. Archival tissue samples of 85 lesions in 58 cases were used. The lesions represented 7 groups: normal esophagus (n = 10), reflux esophagitis (n = 12), Barrett metaplasia (n = 21), Barrett low-grade dysplasia (n = 17), Barrett high-grade dysplasia (n = 5), well- or moderately differentiated adenocarcinoma (n = 10), and poorly differentiated adenocarcinoma (n = 10). Apoptotic cells with fragmented DNA were detected by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) method. Monoclonal antibodies against bcl-2 protein were applied using the avidin-biotin complex immunoperoxidase method. The esophagitis group showed many apoptotic cells on the epithelial surface; in the other groups, few apoptotic cells were seen. Weak bcl-2 expression was seen in the basal cells in normal subjects and those with esophagitis. There was overexpression of bcl-2 in 72% of Barrett metaplasia, 100% of Barrett low-grade dysplasia, 25% of Barrett high-grade dysplasia, 40% of well- or moderately differentiated adenocarcinoma, and 20% of poorly differentiated adenocarcinoma. Increased apoptosis in reflux esophagitis may be a protective mechanism counteracting increased proliferation. Inhibition of apoptosis by overexpression of bcl-2 protein occurs early in the dysplasia-carcinoma sequence of Barrett esophagus. The resulting prolongation of cell survival may promote neoplastic progression. Despite the absence of apoptosis, bcl-2 was not widely overexpressed in Barrett high-grade dysplasia and adenocarcinoma, suggesting that cells acquire other ways of avoiding apoptosis as malignancy appears.

Immunoreactivity of canine mammary neoplasms with monoclonal antibody CC49.

Monoclonal antibody (MAb) CC49 binds to human tumour-associated glycoprotein termed TAG-72. CC49 is a second-generation MAb with higher affinity to TAG-72 than the original MAb B72.3. CC49 was applied to 42 samples from different canine mammary tumours, belonging to seven different histopathological types. Immunoreactivity was detected by the use of an avidin-biotin complex immunoperoxidase method. Most sections from all types of mammary neoplasm reacted with this MAb. Normal tissue did not stain or stained only weakly. The results of this study suggest CC49 has selective immunoreactivity for a variety of canine mammary tumours, which seems superior to that reported with MAb 72.3. These results support the proposal for further study of diagnostic and therapeutic uses of CC49 in the management of canine mammary tumours.

Distribution of nitric oxide synthase-containing ganglionic neuronal somata and postganglionic fibers in the rat kidney.

Nitric oxide synthase (NOS)-immunoreactive neurons were identified in the rat kidney by using an antibody against type Ia NOS and the avidin-biotin complex immunoperoxidase method in whole kidneys examined in 100 microns serial sections. The histochemical method for demonstration of the nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) was also used to characterize NOS-containing neurons. All somata showing NOS immunoreactivity also displayed NADPH-d activity. The greatest number of neuronal somata were observed in groups at the wall of the renal pelvis and in the angular space formed by the pole of the renal parenchyma and renal pelvic wall. They were also seen at the renal hilus close to the renal artery and along the interlobar vasculature. The size of the neuronal somata in the 35-day-old rat ranged from 13.6 to 34.8 microns, with a mean size of 21.52 +/- 4.81 microns. Seventy percent, however, ranged in size from 17.8 to 26.8 microns. The shape of the neuronal somata also varied, with the majority having an ovoid or round shape. The distribution of the postganglionic fibers was investigated by means of the camera lucida. Postganglionic fibers projected into the wall of the renal pelvis and/or to the interlobar arteries extending to the arcuate arteries and to the beginning of the afferent arterioles. The NOS-immunoreactive neurons may have a vasodilator and relaxing function on the renal pelvic wall and vasculature. In addition, the presence of NOS-containing nerve fibers in nerve bundles, which are known to have predominantly vasomotor and sensory fibers, suggest that they may have a possible modulatory role on renal neural function.

Oncogene Protein Co-Expression Value of Ha-ras, c-myc, c-fos, and p53 as Prognostic Discriminants for Breast Carcinoma

A refinement of prognostic variables using traditional pathologic markers integrated with oncogene proteins, enzymes, and hormonal factors may enhance the ability to predict for recurrence or survival in patients with mammary carcinoma. Although various oncogenes and oncogene products have been identified in human breast carcinoma, their relationship to disease outcome remains controversial. Using the monoclonal antibodies cS93.1, 9E1.0, F235-1.7.1, and PAb 1801 against each oncogene protein studied, the avidin-biotin complex immunoperoxidase method provided immunohistochemical staining of bound oncogene protein for c-fos, c-myc, Ha-ras, and p53, respectively. Analyses were made on archival pathology tissues of 85 breast cancer patients (stages I, IIA, and IIB). Forty patients (47%) had recurrence of disease; 45 remained free of local-regional or distant disease at mean follow-up of 48 months (range 6-180 months). Molecular biological data were merged with clinicopathologic demographics 1) to determine the frequency of single or co-expression of oncogenes in this patient population; 2) to evaluate the value of these molecular protein markers to predict probability of recurrence; and 3) to determine worth of the studied oncogenes to correlate with traditional clinical pathologic parameters and overall survival. In this study, oncogene expression had statistical correlation for recurrence with increasing co-expression: one oncogene 17.2%, two oncogenes 56.3%, three or four oncogenes, 100% (p = 0.001). Increasing oncogene or co-oncogene expression correlated with statistically significant reduction in disease-free and overall survival; with no expression of oncogenes, disease-free survival was 30 (SE +/- 5.7) months and overall survival was 56.4 (SE +/- 4.57) months. With expression of three oncogenes, disease-free survival was 12 (SE +/- 1.23) months (p = 0.0018) and overall survival was 23.4 (SE +/- 3.38) months (p = 0.0025). In univariate Wilcoxon analysis, oncogene expression was the most significant variable to determine survival (p = 0.035); in multivariate analysis, age and oncogene co-expression each emerged as the most significant variables for overall survival. For the proportional hazards regression model, oncogene co-expression was significant (p = 0.0104, risk-ratio 1.914) and correlated with age and tumor size as significant variables. Ha-ras and c-fos both emerged as important individual oncogene proteins to affect survival (p = 0.0925, risk-ratio 3.517 and p = 0.025, risk-ratio 4.214, respectively). The proto-oncogene c-myc and the antitumor suppressor gene p53 did not have significant effects as individual oncogenes to influence survival. Approximately one fifth of the breast cancer patients in this analysis (disease-free and recurrent) expressed only a single oncogene marker (c-fos, c-myc, Ha-ras, or p53); one quarter of patients with recurrent disease expressed only one oncogene protein. Single oncogene expression did not possess independent prognostic significance for prediction of recurrence. Further, p53 mutations did not function as independent correlates for prognosis. The co-expression of the studied proto-oncogenes (c-myc, Ha-ras) and the nuclear transcriptional protein (c-fos) functioned as a strong prognostic correlate for recurrence and survival; the effect of individual oncogenes to predict survival was greatest for Ha-ras and c-fos. Immediate or early co-expression of three oncogene proteins in neoplastic transformation endowed cells of invasive carcinoma with an aggressive phenotype. This aggressive phenotype was evident in a small percentage of the studied population (11%) and predicted adverse disease-free and overall survival. These findings suggest that oncogene co-expression possesses significant prognostic and potential therapeutic value; incorporation of this molecular technology into future prospective randomized trials is advisable.

Immunoreactivity of canine transitional cell carcinoma of the urinary bladder with monoclonal antibodies to tumor-associated glycoprotein 72.

Tumor-associated glycoprotein 72 (TAG-72) is a large, high molecular weight, mucinlike antigen that is expressed in a wide variety of human carcinomas. Three different TAG-72 monoclonal antibodies (MAbs), designated B72.3, CC49, and CC83, were applied to the following archived samples from the dog: 1) 51 transitional cell carcinomas of the urinary bladder, 2) 15 hyperplastic/inflamed urinary bladders, and 3) eight normal urinary bladders. Immunoreactivity was detected with an avidin-biotin complex immunoperoxidase method. Fifty-three percent (27/51) of transitional cell carcinomas were positive (> or = 5% staining) for MAb B72.3. MAb B72.3 staining of these transitional cell carcinomas did not statistically correlate with any of the examined features of malignancy, including histologic grade, clinical stage, DNA ploidy, or presence of vascular/lymphatic invasion. In regard to the staining of transitional cell carcinoma by the other two TAG-72 antibodies, 53% (27/51) of the samples were positive for MAb CC83 and 63% (32/51) were positive for MAb CC49. The finding that similar populations of neoplastic urothelial cells in serial sections from the same neoplasm stained with all three TAG-72 antibodies supports the hypothesis that an antigen similar to TAG-72 was expressed in canine transitional cell carcinoma. None of the normal urinary bladders nor any of the hyperplastic/inflamed urinary bladders stained with any of the three TAG-72 antibodies tested. The results of these studies demonstrated that the staining of canine transitional cell carcinoma with all three TAG-72 antibodies was specific for neoplastic urothelial cells and that an antigen similar to TAG-72 was expressed.

Immunoreactivity of Canine Epithelial and Nonepithelial Neoplasms with Monoclonal Antibody B72.3

Monoclonal antibody (MAb) B72.3, which binds to a human tumor-associated glycoprotein termed TAG-72, was applied to a wide range of epithelial and nonepithelial neoplasms from dogs. Immunoreactivity was detected by the use of an avidin-biotin complex (ABC) immunoperoxidase method. A variety of epithelial neoplasms, but none of the nonepithelial neoplasms, were positive (> or = 5% staining) for MAb B72.3. MAb B72.3 stained 100% (4/4) of gastric, 100% (6/6) of intestinal, 50% (2/4) of pancreatic, and 80% (4/5) of rectal adenocarcinomas but only 20% (1/5) of squamous cell carcinomas and 20% (1/5) of complex tubular mammary gland adenocarcinomas. None of the hepatocellular carcinomas and perianal and sebaceous gland adenocarcinomas stained. Most types of benign epithelial neoplasms did not stain, except for 75% (6/8) of rectal adenomas and 25% (1/4) of squamous cell papillomas. Normal gastrointestinal mucosa adjacent to and entrapped in neoplasms did stain with MAb B72.3. None of the benign and malignant nonepithelial neoplasms of mesenchymal, neuroendocrine, or lymphohematopoietic tissue origin stained. The results of this study demonstrate that MAb B72.3 has selective immunoreactivity for adenocarcinomas, especially those arising from the digestive tract; however, limited immunoreactivity was observed for other types of carcinomas and benign epithelial neoplasms and for normal gastrointestinal mucosa in the dog.