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PubMed2006

Hypermethylation of the p16 gene promoter in pterygia and its association with the expression of DNA methyltransferase 3b.

PURPOSE: A pterygium has long been considered as a degenerative condition. After p53 protein was found to be abnormally expressed in the epithelium, researchers suggested that a pterygium may be a tumor, but additional evidence is required to support this hypothesis. Aberrant methylation of the p16 gene (CDKN2A) promoter and resultant gene silencing play important roles in the pathogenesis of many types of human cancers. The purpose of this study was to investigate hypermethylation of the p16 promoter in pterygia and the relationship between this hypermethylation and the expression of p16 and DNA methyltransferase 3b (DNMT3b) proteins. METHODS: We studied the methylation status of p16 and the expression of p16 and DNMT3b proteins by performing methylation-specific polymerase chain reaction and immunohistochemistry, respectively, in specimens of 129 pterygia and 16 normal conjunctiva. The results were statistically analyzed. RESULTS: Hypermethylation of the p16 gene promoter was detected in 21 (16.3%) of 129 pterygial specimens. Among them, 46 (35.7%) were positive for p16 protein expression, and 83 (64.3%) were negative. Staining for p16 was limited to the nuclei of the epithelial layer. We observed a significant reverse correlation between hypermethylation of the p16 promoter and the expression of p16 protein (p=0.006). Thirty-eight (29.5%) pterygial specimens were positive for DNMT3b protein expression, and 91 (70.5%) were negative. DNMT3b staining was limited to the nuclei of the epithelial layer. A significant correlation was found between hypermethylation of the p16 promoter and the expression of DNMT3b protein (p<0.001). CONCLUSIONS: The p16 gene promoter was hypermethylated in pterygia, and this hypermethylation was strongly linked to expression of the positive expression of DNMT3b protein and to the suppression of p16 protein. These data provided molecular evidence that methylation occurs in pterygia and that it may play a role in the their development.

Aged↗
PubMed1993

Two-dimensional crystallization of the light-harvesting complex from Rhodospirillum rubrum.

Homogeneous detergent-solubilized B873 light-harvesting complexes from a carotenoid-less mutant of the purple non-sulfur bacterium, Rhodospirillum rubrum G9, were reassembled spontaneously into two-dimensional (2D) hexagonal arrays during extensive and controlled dialysis. As the complexes contain only 1 to 2 mol phospholipid per mol alpha beta dimer, the arrays formed by a self assembly process are primary due to protein-protein interactions. The hexagonal lattices were analyzed by negative stain electron microscopy and digital image processing. They exhibited a unit cell size of 12.3 nm, in close agreement with the particle diameter of the active photo-unit in native chromatophore membranes. The unit cell contains a central 5 nm stain-filled depression, embraced by a ring with an outer diameter of 10 nm.

Crystallization↗
PubMed1997

[Detection of human papilloma virus DNA and expression of p53 protein in patients with head and neck cancer].

We investigated the p53 expression and the presence of HPV DNA in 90 patients with squamous cell carcinomas (SCCs) of the head and neck and the relation to clinicopathological parameters and patients' prognosis. Immunohistochemical analysis of p53 protein was conducted by using monoclonal anti-p53 antibody, clone 1801 and clone 240. The relationship between the overexpression of p53 and the duration of survival of patients was analyzed. The polymerase chain reaction (PCR) was carried out with consensus primers capable of detecting HPV16, 18, 31, 33, 52b and 58. In situ hybridization was performed in the mesopharyngeal carcinoma to confirm the presence of HPV genomes in cancer cells with a wide-spectrum cDNA probe capable of detecting HPV6, 11, 16, 18, 30, 31, 33, 35, 45, 51, 52. Forty-five tissue samples (50%) were immunohistochemically positive for p53. T-category, N-category, primary site of tumor, clinical stage and tumor differentiation did not correlate with p53 expression. Our finding that p53 overexpression occurred in 50% of head and neck tumor samples is similar to the frequency of p53 overexpression reported for both lung and esophageal cancer. The common risk factor is the same in these neoplasms, and therefore it is not surprising to find a similar percentage of p53 overexpression. The prevalence of metastasis was higher in the patients with p53-positive staining than in those with p53-negative staining (p < 0.10). Analysis of cumulative survival rates of patients by the Kaplan-Meier method showed a close correlation between p53 expression and survival time. The survival differences according to p53 immunostaining were significant (p 0.05). Our results indicate that p53 immunohistochemical evaluation may be useful as one of the new prognostic parameters in head and neck cancer patients. The HPV genomes were detected in 9 of 90 patients (10.0%); 8 of 9 patients with mesopharyngeal cancer and one with maxillary cancer, namely, 29.6% of mesopharyngeal cancers and 6.7% of maxillary cancer contained HPV DNA sequences. Seven of 8 patients had SCCs of tonsil origin. Almost all of the HPV infections in our study occurred in patients with mesopharyngeal cancer, and it has been suggested that this anatomic subsite may be more frequently infected by HPV than other sites within the head and neck region. Among the 27 patients with mesopharyngeal cancer, HPV DNA-positive patients experienced a higher incidence of complete remission than HPV DNA-negative patients (87.5% vs. 26.3%, p < 0.05).

Aged↗
PubMed2000

Sentinel node dissection and ultrastaging in squamous cell cancer of the vulva.

OBJECTIVE: The aim of this study was to evaluate the findings when pathologic ultrastaging techniques are applied in conjunction with sentinel node dissection in patients with vulvar cancer. METHODS: Patients with squamous cell cancer of the vulva underwent intraoperative lymphoscintigraphy following intradermal injection of 99mTc-labeled sulfur colloid at the site of the primary tumor. Isosulfan blue dye was also injected at the tumor site to facilitate identification of the sentinel node in the groin. Following removal, the sentinel node was then bisected and examined in the standard manner using hematoxylin and eosin staining. Negative nodes were subjected to additional ultrastaging evaluation with serial sectioning and immunohistochemical staining. RESULTS: Nine patients with 10 primary tumors underwent radical local excision of the primary tumor and sentinel node dissection of the groin. Sentinel nodes were identified and removed in all patients. One node was positive by conventional staining; the remainder were all negative. Of these negative nodes, 2 were found to be positive for micrometastases on serial sectioning and immunohistochemical staining. Therefore 2 of 3 positive nodes were not detected using conventional histologic techniques. CONCLUSION: Sentinel node dissection appears to be technically feasible in patients with vulvar cancer. Pathologic ultrastaging combined with sentinel node dissection appears to be highly sensitive for detecting subclinical micrometastases in the regional lymphatics. This technique potentially provides a more accurate assessment with less surgical morbidity than conventional inguinalfemoral lymphadenectomy.

Aged↗
PubMed1986

The microvascular localization of laminin and fibronectin in scleroderma muscle by immune electron microscopy.

Immune electron microscopy using a peroxidase-antiperoxidase (PAP) technique was done on muscle biopsies from patients with scleroderma (PSS), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and normal controls, in order to localize laminin and fibronectin in the muscle capillary basement membrane (BM). Three patterns of staining were noted for the 2 antigens: in/out, diffuse and negative. Staining patterns in PSS biopsies were markedly different from both the normal and disease controls. For laminin the expected in/out pattern was rare in PSS, with virtually all biopsies being either diffuse or negative. In the case of fibronectin, the staining patterns were of equal frequency in PSS, whereas in the control groups diffuse staining was rare. These findings suggest that in addition to the morphologic abnormalities previously noted in PSS muscle capillary BM, there is also an alteration in the distribution of specific BM components in PSS.

Arthritis, Rheumatoid↗
PubMed2000

Pleural fluid cytology of Hodgkin's disease: cytomorphologic features and the value of immunohistochemical studies.

Two cases in which Hodgkin's disease (HD) was cytologically diagnosed in pleural effusions are presented. The presence of Reed-Sternberg (R-S) cells was confirmed by positive staining for both CD15 and CD30, and negative staining for leukocyte common antigen. In addition, the differential diagnosis of HD in effusion cytology is presented, including look-alikes of R-S cells that can potentially lead to an incorrect diagnosis. To the best of our knowledge, we believe this is only the second cytologic report of HD diagnosed in a pleural effusion using immunohistochemistry to confirm the diagnosis.

Adult↗
PubMed1986

Histiocytoid carcinoma: a variant of breast cancer.

The history of a patient presenting with metachronous bilateral breast cancer displaying histiocytoid features is reviewed. Although regional metastases were noted, this patient has not demonstrated an aggressive systemic disease pattern. In the past, histiocytoid breast cancer has been classified as either a lipid-rich carcinoma or as a variant of lobular carcinoma. However, histiocytoid carcinoma should be considered a distinct entity. Unlike the lipid-rich carcinomas, this tumor stained strongly for mucin. Immunoperoxidase staining indicated strong positively for CEA and negative staining for alpha-lactalbumin. There is suggestive evidence of a relationship between histiocytoid breast carcinoma and breast cancers of apocrine origin. Controversy remains and further evaluation is needed to elucidate the histiogenesis and biological potential of this neoplasm.

Axilla↗
PubMed2005

Malignant stromal tumor, so called "gastrointestinal stromal tumor", with rhabdomyomatous differentiation occurring in the gallbladder.

Gastrointestinal stromal tumors (GISTs) constitute the largest category of primary nonepithelial neoplasms of the gastrointestinal tract. It is extremely rare that this tumor occurs in the bile tract, and only a few cases have been reported. Immunohistochemically, the tumor cells revealed a phenotype similar to Cajal cells, occasionally with differentiation to smooth muscle cells or neural cells. We present a case of malignant stromal tumor similar to GISTs with rhabdomyomatous differentiation of the gallbladder in a 68-year-old woman. The resected tumor was predominantly composed of spindle cells with rhabdomyomatous differentiation. Immunohistochemical study revealed diffuse staining of tumor cells using vimentin despite negative staining for desmin or S-100. This indicated a mesenchymal origin of the cells without smooth muscle or neuronal differentiation. Myoglobin-positive cells, in which phosphotungstic acid hematoxylin staining revealed cross striations of the cytoplasm, suggested rhabdomyomatous differentiation. Diffuse positivity for KIT in the cells suggested that the pathogenesis of this tumor may resemble that of GIST. The tumor may have derived from a mesenchymal stem cell that had undergone partial rhabdomyomatous differentiation.

Aged↗
PubMed2006

Cryopreservation of bull spermatozoa alters the perinuclear theca.

The perinuclear theca (PT) is involved in several important sperm functions leading to fertilization. The objective of this study was to investigate the effect of cryopreservation of bull spermatozoa on the integrity of the PT and the relationship between PT integrity and semen characteristics. Semen from seven bulls was evaluated before and after cryopreservation, comparing the integrity of the plasma membrane (hypo-osmotic test), percentage of live and dead spermatozoa (triple stain), acrosome integrity (triple stain) and the integrity of the PT (negative stain by electron microscopy). Cryopreservation of bull semen caused substantial damage to the PT; the proportion of spermatozoa with a damaged PT was 15.2% versus 52.5% (P<0.05) in fresh versus frozen-thawed spermatozoa, respectively. Furthermore, on average, 67.4% (range, 64-72%) of fresh spermatozoa were live, compared to 53.1% (range, 49-58%) for frozen-thawed spermatozoa; there was an inverse correlation between the percentage of live spermatozoa and the percentage with damage to the PT. Although 59.1% of frozen-thawed spermatozoa had an intact acrosome, only 43.7% of them still remained alive. In frozen-thawed semen, there was a high correlation (r=0.69) between live spermatozoa with an intact acrosome and spermatozoa that maintained an intact PT. In conclusion, freezing/thawing of bull spermatozoa altered the PT and maintaining PT integrity may be necessary to maintain acrosome integrity.

Acrosome↗
PubMed1996

Hyalinizing trabecular carcinoma of thyroid gland.

We describe two cases of encapsulated thyroid tumours which displayed the classic morphological features of hyalinizing trabecular adenoma. In addition, both were characterized by focal invasion of the capsule and of thin-walled capsular blood vessels. Positive immunohistochemical staining of tumour cells for thyroglobulin and negative staining for calcitonin, chromogranin and CEA allowed distinction from medullary carcinoma. Electronmicroscopy revealed groups of tumour cells, surrounded by abundant basement membrane type material. Occasional tumour cells contained abundant cytoplasmic intermediate filaments. A flow cytometric analysis revealed one tumour to have a diploid DNA pattern and the other to be DNA aneuploid. These cases illustrate that a malignant variant of hyalinizing trabecular adenoma, namely hyalinizing trabecular carcinoma, exists. Hyalinizing trabecular tumours of the thyroid should not be considered uniformly benign lesions. As with follicular neoplasms, multiple sections from the capsule should be examined histologically in order to assess the presence or absence of capsular and/or vascular invasion.

Carcinoma↗
PubMed1994

Immunohistochemical analysis of cutaneous malignant melanoma: comparison of S-100 protein, HMB-45 monoclonal antibody and NKI/C3 monoclonal antibody.

Immunohistochemical analysis was carried out on 144 formalin fixed paraffin embedded cutaneous melanomas to ascertain the value of 3 different immune markers. The sensitivity and staining patterns regarding intensity and distribution, as well as correlation to pigment content, cell type, surface ulceration and host response was noted. The stains used were monoclonal HMB-45 (Dako product No M634), NKI/C3 antibodies (Biogenex product No MU077) and polyclonal rabbit anti S-100A protein (Dako product No L1845). Of the lesions tested, 63 were malignant melanoma with an adjacent component of superficial spreading type, 61 were malignant melanoma with no adjacent component, 2 were malignant melanoma with adjacent lentigo maligna, 8 were in situ with 7 superficial spreading melanoma and one lentigo maligna (HMF) and 10 were metastatic melanoma. All 144 lesions stained for S-100 (100% sensitivity). One hundred and thirty-seven stained for NKI/C3 (95%); 132 stained for HMB-45 (92% sensitivity). S-100 was the most sensitive marker and stained tumor cells diffusely. With HMB-45 and NKI/C3, though marginally less sensitive, staining was stronger and patchy. In addition, NKI/C3 showed a tendency for peripheral (membrane) staining. HMB-45 staining was directly proportional to the pigment content, with stronger staining of radial growth phase melanoma and negative staining of those lesions where pigment content was minimal or absent. Also, with HMB-45 a decrease in staining intensity with depth or vertical growth phase was observed. There was no relationship to cell type with HMB-45 but with NKI/C3, 5 out of the 7 that failed to stain showed spindle cell differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1999

Secretory carcinoma of the breast in a nine year old boy.

Carcinoma of the breast is very rare in childhood and is exceedingly rare in boys. Secretory carcinoma, a distinctive and rare variant of breast carcinoma is for some unknown reason the commonest type seen in children. To our knowledge there have been only four previous reports in boys under ten years old. We report the first case in Australia of this unusual tumor in a nine year old boy. The child presented with a subareolar nodule 12 mm in its greatest dimension. High resolution sonography showed a well defined hypoechoic nodule. Histology revealed classical features of secretory carcinoma with circumscribed, pushing margins, except for one site of invasion. The tumor displayed the typical cribriform and microcystic pattern with PAS positive, diastase resistant secretions, and lack of pleomorphism and mitotic activity. Tumor cells showed positive staining with S100 and polyclonal CEA and negative staining for estrogen and progesterone receptors. Although, because of its rarity, the natural history of this tumor is not well documented and optimal management is uncertain, prognosis happens to be excellent as these tumors behave in an indolent manner, both in children and in adults.

Biomarkers, Tumor↗
PubMed2000

Intraepidermal Merkel cell carcinoma with no dermal involvement.

Cutaneous Merkel cell carcinoma (MCC) typically involves the dermis. Less than 10% of MCC have epidermal involvement. Only one MCC confined exclusively to the epidermis has been previously reported but was not recognized until the lesion recurred with typical MCC in the dermis. We present a case of a wholly intraepidermal pagetoid MCC without dermal involvement in a 74-year-old man with a 2.0-cm solitary verrucous papule on the left index finger. The initial biopsy and complete excision specimens showed marked epidermal hyperplasia, focal prominent squamous cell atypia, and MCC with florid pagetoid spread through the epidermis. There was no evidence of tumor within the dermis. The pagetoid MCC tumor cells showed diffuse cytoplasmic staining with antibodies to cytokeratin 20, and negative staining for chromogranin, neurofilament, S-100, vimentin, HMB45, leukocyte common antigen, and CD3. The cell of origin of MCC is still debated. The existence of an entirely intraepidermal variant of MCC would lend support to the view that MCC is a neoplastic expression of Merkel cells in at least some cases. Dermal-based MCC is a high-grade primary cutaneous neoplasm, but MCC confined exclusively to the epidermis may have a better prognosis.

Aged↗
PubMed1993

Meningioma-like tumor of the skin. An ultrastructural and immunohistochemical study.

Three unusual cutaneous tumors are described along with ultrastructural and immunohistochemical studies. All lesions were asymptomatic red-brown papulonodules. Light microscopic examination revealed a whorled configuration of spindle-shaped cells, some concentrically arranged around blood vessels. Immunohistochemical panels exhibited positive staining only with antibody to vimentin and negative staining with antibodies against S-100 protein, muscle markers, cytokeratin, epithelial membrane antigen, Leu 7, type IV collagen, and factor XIIIa, ruling out obvious nevomelanocytic, nerve sheath, meningothelial, smooth muscle, and perithelial differentiation. Electron microscopic examination demonstrated cells producing poorly formed collagen fibrils, sparse collagen fibers, and possessing occasional ill-defined intercellular junctions between their elongated cell processes. This rare tumor is considered to be either an immature fibrohistiocytic or possibly a nerve sheath neoplasm with striking similarities to so-called canine hemangiopericytoma. Because the prominent whorled pattern was reminiscent of meningioma, the lesion was referred to as meningioma-like tumor of the skin.

Actins↗
PubMed1985

Histogenesis of post-mastectomy angiosarcoma--an ultrastructural study.

An example of post-mastectomy angiosarcoma was studied by electron microscopy to determine its histogenesis. Unequivocal evidence of endothelial differentiation was found in well and poorly differentiated areas. Positive staining for factor VIII related antigen and negative staining for epithelial membrane antigen further confirmed that the tumour was a true angiosarcoma rather than a peculiar form of carcinomatous metastasis.

Blood Vessels↗
PubMed2006

Oncocytic non-functioning endocrine tumor of the pancreas.

Herein is presented the case of a malignant non-functioning endocrine tumor of the pancreas with oncocytic features, and a discussion on the high incidence of malignancy in oncocytic endocrine pancreatic tumors. The patient was a 65-year-old woman who showed no paraneoplastic symptoms produced by functioning pancreatic endocrine tumors. The primary tumor was located in the body and tail of the pancreas, and had metastasized to the liver. Tumor cells were arranged in a ribbon-like or trabecular pattern and had an abundant eosinophilic cytoplasm containing numerous mitochondria and neurosecretory granules. The cytoplasm of the tumor cells was intensely stained with an antimitochondrial antigen antibody. Most tumor cells stained positively with Grimelius stain and for chromogranin A. Some tumor cells also stained for synaptophysin. However, the tumor cells negatively stained for hormones such as insulin, glucagon, somatostatin, gastrin, vasoactive intestinal peptide and pancreatic polypeptide, for serotonin, and for pancreatic enzymes such as amylase and trypsin. Analysis of 18 oncocytic pancreatic endocrine tumors, consisting of those reported previously and that in the present case, suggests that the high incidence of malignancy in oncocytic endocrine tumors is associated with the high incidence of non-functioning endocrine tumors among them, most of which are malignant.

Adenoma, Oxyphilic↗
PubMed1999

Pretreatment in a high-pressure microwave processor for MIB-1 immunostaining of cytological smears and paraffin tissue sections to visualize the various phases of the mitotic cycle.

In many pathology laboratories, both microwave ovens and pressure cookers are used for pretreatment of cytologic smears and paraffin sections to allow MIB-1 staining. For both methods there are two problems. First, the results cannot be used for quantitation because standardization is impossible. Second, the staining results are often suboptimal, resulting in negative staining of cells in the G(1)- and S-phases. When pretreatment is performed in a microwave processor, allowing microwave heating under pressure, precise temperature monitoring becomes possible. In addition, the importance of the pH of the buffer was studied using a test battery series. Optimal staining is achieved at a temperature of 115C, 10 min, pH 6. This method proved to be highly reproducible. Because the immunostaining results are optimal, the various phases of the cell cycle can be defined in the sections and smears. In addition, the perinucleolar staining of the late G(1)-phase is optimally visualized and nuclei of the stable pKi-67 pathway can be identified. Under suboptimal conditions, in particular, the number of cells in the late G(1)-phase are underestimated in the MIB-1 counts.

Antigens, Nuclear↗
PubMed2002

[A modified method for isolating and culturing peritoneal mesothelial cells in rats].

OBJECTIVE: To establish a culture model of peritoneal mesothelial cells in vitro separated by infusing the digestive fluid in the rat abdominal cativity. METHODS: Peritoneal mesothelial cells (PMC) were digested with 0.125% trypsin-0.01% EDTA Na2 in the abdominal cavity. We collected coelio-perfusate and cultured it. The isolated cells were identified by the phase contrast microscope, electron microscope and immunohistochemical analysis. RESULTS: The confluent cells showed a uniform cobblestone-like appearance under the phase contrast microscope; numerous surface microvilli could be found under the electron microscope and the purity of the cells was over 95%. Immunohistochemical studies revealed a positive staining for cytokeratin and vimentin, but a negative staining for Factor VIII associated antigen and CD45. CONCLUSION: The model of rat peritoneal mesothelial cells has been established successfully and it will provide an experimental basis of studying the fibrosis in peritoneal dialysis.

Animals↗