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PubMed1991

Differentiation dependent expression of P-glycoprotein in the normal and neoplastic human kidney.

Adult renal cell carcinoma (RCC) is clinically resistant to chemotherapy. However, in nephroblastoma (NBL) chemotherapy has increased survival dramatically. We studied the P-glycoprotein (P-gp) expression of 18 RCC and 9 NBL as well as 1 benign renal adenoma and fetal renal tissue using three different monoclonal antibodies (MRK-16, C-219, JSB-1). P-gp was found positive with all three antibodies in 12/18 RCC, while only 2 tumors were completely negative. Staining varied with respect to intensity and number of positive cells [5%-90%]. Intense staining was seen at the apical side of malignant tubules in well differentiated parts of RCC and in tubular structures of the benign renal adenoma. Poorly differentiated parts of the tumors showed less staining. In NBL blastemal parts were negative. In 4/8 specimens showing focal epithelial differentiation, however, the luminal side of more differentiated tubular structures did stain, strongly resembling P-gp staining in the developing fetal human kidney. These results indicate that P-gp expression in normal (fetal) human kidney as well as in benign and malignant tumors derived from this organ depends on the degree of differentiation of tubules, which may have implications for chemotherapy sensitivity in both malignant tumors.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Loss of tumor marker-immunostaining intensity on stored paraffin slides of breast cancer.

BACKGROUND: We previously observed decreased p53 immunostaining over time in paraffin-embedded sections of ductal carcinoma in situ of the breast of women; these sections had been stored on slides at room temperature. This observation suggests that slide storage adversely affects p53-immuno-staining intensity and could result in spurious negative staining for p53 in patient samples. PURPOSE: The goals of this study were to determine the time course and factors influencing loss of p53 immunoreactivity and to investigate whether a similar loss of reactivity occurs with other antigens commonly used to study breast cancer. METHODS: Serial sections cut from 12 formalin-fixed, paraffin-embedded, p53-positive invasive ductal carcinomas of the breast were stored on slides at room temperature or at 4 degrees C, with or without an additional paraffin coating, for 2, 4, 8, or 12 weeks. For each case, freshly cut slides from the same block (day 0) and stored slides were simultaneously stained for p53 by use of an automated immunostainer. Slides cut from formalin-fixed, paraffin-embedded breast carcinomas and stored for 12 weeks were also stained for factor VIII-related antigen (n = 12), estrogen receptor (ER) (n = 9), and Bcl-2 protein (n = 9). The staining intensity of all slides was assessed by visual microscopic examination and was also quantified by image analysis. Quantitative results were expressed as a percentage (mean +/- standard error) of the staining intensity on day 0. Data were analyzed by the Friedman Repeated Measures Analysis of Variance on Ranks, with statistical significance set at two-sided P < .05. RESULTS: The intensity of p53 staining decreased over time in nine (75%) of the 12 cases studied. In three (or 25% of all cases studied) of the nine cases that showed decreased p53 staining, slides stored for 12 weeks were scored as p53 negative. Antigen loss on slides stored at 4 degrees C was significantly less than that on slides stored at room temperature at all time points (all P < .05). At 12 weeks, the average staining intensity of slides stored at 4 degrees C was 33.2% +/- 9% of that on day 0 compared with 8.4% +/- 3% of that on day 0 for slides stored at room temperature (P < .001). Paraffin coating of the sections did not significantly diminish antigen loss at either room temperature or 4 degrees C, except for slides stored at room temperature for 12 weeks. The intensity of factor VIII staining decreased in nine of 12 cases (average staining intensity, 37.3% +/- 6% of that on day 0 at 12 weeks; P = .0001). The intensity of ER and Bcl-2 staining decreased in all nine cases studied at 12 weeks (average staining intensity, 14.0% +/- 6% and 21.0% +/- 4% of that on day 0, respectively; P = .0001 for each). CONCLUSIONS AND IMPLICATIONS: Slide storage, particularly at room temperature, results in substantial loss of p53 reactivity, with some p53-positive cases becoming p53 negative after 12 weeks of storage. Substantial loss of immunoreactivity for factor VIII, ER, and Bcl-2 occurs on slides stored at room temperature for 12 weeks. Storage of unstained slides for up to 12 weeks may lead to false-negative immunostaining for p53 and other antigens.

Biomarkers, Tumor↗
PubMedDate not supplied

Enolase isoenzymes in benign and malignant melanocytic lesions.

The distributions of the alpha, beta and gamma subunits of enolase were studied in 34 melanocytic lesions using the peroxidase-antiperoxidase (PAP) technique. Melanocytes in benign neoplasms stained moderately strongly for both alpha enolase and gamma enolase but staining for beta enolase was consistently negative. Staining for alpha enolase was always stronger than that for gamma enolase. There are some indications that with increasing degrees of dedifferentiation there was a reduction of gamma enolase activity, least differentiated cells being completely negative. Ocular melanomata showed similar changes to those seen in the skin. The enolase isoenzyme composition of tumour extracts was assessed biochemically on a limited number of cases and these results are discussed in relationship to the immunohistochemical studies.

Humans↗
PubMed1997

Anticytokeratin antibody 34 beta E12 staining in prostate carcinoma.

Anticytokeratin antibody 34 beta E12 is advocated as an immunohistochemical stain for discriminating benign and malignant lesions of the prostate. Positive staining with 34 beta E12 is said to identify benign lesions, whereas negative staining is said to help substantiate a diagnosis of carcinoma. It is further claimed that 34 beta E12 does not stain prostate carcinoma. The studies leading to these conclusions used hematoxylin-eosin-stained sections of primary prostate lesions as controls. Although the cytokeratin content of a few cell lines of metastatic prostate carcinoma has been investigated, the 34 beta E12 immunohistochemical staining of metastatic prostate carcinoma has not been evaluated. If 34 beta E12 positivity is present only in benign prostate cells, then metastatic prostate carcinoma cells should be uniformly negative with this stain. In 14 cases of moderate and high-grade prostate cancer with metastases to lymph nodes, we found 34 beta E12 positivity in 6 (43%) of 14 metastases and in 7 (54%) of 13 primary tumors. Our findings of 34 beta E12 staining in primary and metastatic moderately and poorly differentiated prostate carcinoma differ from those reported in the literature for well-differentiated prostate carcinoma. We urge caution in the use and interpretation of 34 beta E12 staining for the diagnosis of primary and metastatic prostate carcinoma.

Adenocarcinoma↗
PubMedDate not supplied

Immunohistochemical detection of mutant p53-suppressor gene product in patients with breast cancer: influence on metastasis-free survival.

Overexpression of p53-protein appears to be a common event in primary breast cancer. It has been proposed that the presence of elevated levels of this protein may be an independent prognostic factor and may be important for the ability of a tumor to metastasize. This study was performed to evaluate the influence of immunohistochemically detectable mutant p53-protein on metastasis-free survival of patients with breast cancer. Immunohistochemistry was performed on 117 paraffin-embedded biopsy specimens of consecutive patients with stage T1-T4 breast cancer, using a monoclonal antibody against p53 suppressor gene product. 29 (24.8%) specimens showed positive staining, whereas in 88 (75.2%) a negative staining reaction for p53 was found. Comparing time intervals to diagnosis of metastasis, using Kaplan-Meier curves, Log-Rank test revealed no significant differences in metastasis-free survival between p53 positive and negative patients (P = 0.32), whereas statistically significant differences were noted for tumor stage (P < 0.01), nodal status (P < 0.01), histological grading (P < 0.01) and estrogen receptor status (P = 0.03). Mutant p53-protein, as detected by immunohistochemistry in paraffin embedded tumor tissue, does not appear to influence metastasis-free survival in patients with breast cancer.

Biomarkers, Tumor↗
PubMed1994

p53 immunohistochemical analysis in breast cancer with four monoclonal antibodies: comparison of staining and PCR-SSCP results.

The expression of p53 protein was examined in a series of 136 primary breast carcinomas, 106 of which were analysed with a panel of four monoclonal antibodies (MAbs 1801, 240, DO7 and DO1). p53 expression was detected with at least one antibody in 40 tumours (38%), whereas only 15 tumours (14%) were positive with all four antibodies. Some variability in the immunostaining could be observed depending on the antibody used. This was noticeable both for the number of positive cells within a section and for the intensity of staining. We therefore selected a panel of 17 tumour sections (nine were highly positive, three with medium to low staining and five with low to negative staining), which we analysed by polymerase chain reaction-single-strand conformation polymorphism analysis (PCR-SSCP) for the presence of a p53 mutation at the molecular level. Mutations were identified in 15 cases. Therefore the proportion of p53-stained cells does not seem to be an exact representation of the number of cancer cells bearing a mutation within a tumour. A statistically significant correlation was observed between p53 expression, regardless of the number of positive antibodies, and grade III disease (P < 0.0001), oestrogen (P < 0.0001) or progesterone receptor negativity (P = 0.0061), increased Ki 67 index (P = 0.0018), epidermal growth factor receptor (EGFR) positivity (P = 0.0076) and aneuploidy (P = 0.037). No correlation was observed with tumour size or lymph node involvement. In univariate analysis p53 expression was not correlated with disease-free survival, in contrast to the classical prognostic parameters, which were statistically correlated. In this series p53 expression was not a marker of early recurrence.

Antibodies, Monoclonal↗
PubMed2001

Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors.

This study details the profile of 13 cell surface cluster differentiation markers on human reserve stem cells derived from connective tissues. Stem cells were isolated from the connective tissues of dermis and skeletal muscle derived from fetal, mature, and geriatric humans. An insulin/dexamethasone phenotypic bioassay was used to determine the identity of the stem cells from each population. All populations contained lineage-committed myogenic, adipogenic, chondrogenic, and osteogenic progenitor stem cells as well as lineage-uncommitted pluripotent stem cells capable of forming muscle, adipocytes, cartilage, bone, fibroblasts, and endothelial cells. Flow cytometric analysis of adult stem cell populations revealed positive staining for CD34 and CD90 and negative staining for CD3, CD4, CD8, CD11c, CD33, CD36, CD38, CD45, CD117, Glycophorin-A, and HLA DR-II.

Aged↗
PubMed1995

Characteristics of nine newly derived mesothelioma cell lines.

This report characterizes nine new cell lines derived from patients with malignant pleural mesothelioma. The lines were initiated between July 1990 and July 1992 from solid tumors (5 lines) or effusions (4 lines) and had proliferated for a period of at least 2 months without senescence. They were characterized by cell size, doubling time, immunohistochemical analyses, electron microscopy, and chromosomal karyotyping. Growth factor/cytokine elaboration was determined using enzyme-linked immunoassays. The established lines were similar in morphology to their parent tumor (ie, epithelial or sarcomatoid). Cell sizes ranged from 59 to 81 microns, and the doubling times varied from 31 to 65 hours. The lines stained with cytokeratin and showed expected negative staining for adenomarkers including B72.3 and carcinoembryonic antigen. All cell lines exhibited aneuploidy, with modal chromosome numbers between 40 and 81 and had multiple chromosomal aberrations. Significant production of granulocyte-monocyte colony-stimulating factor, leukemia inhibitory factor, platelet-derived growth factor, and interleukin-6 was seen. These new cell lines derived from human mesotheliomas can now be used to aid in the design of innovative treatment strategies.

Adult↗
PubMed1992

Generalized inflammation during peritonitis evidenced by intracutaneous E-selectin expression.

E-selectin is a human endothelial leukocyte adhesion molecule which is expressed on endothelial cells after exposure to inflammatory mediators and which is known to be involved in the adhesion of polymorphonuclear cells to the endothelium in vitro. Data on E-selectin expression in vivo are limited. In the present report, we studied the expression of E-selectin in skin biopsies from patients with peritonitis due to a perforation of the gastrointestinal tract. Substantial E-selectin expression was observed on the vasculature of the skin from six out of eight of these severely ill patients. Skin obtained from healthy individuals stained negative, or showed a faint patchy staining in 30% of biopsies tested. These results provide evidence that E-selectin expression was induced at a distance from the primary inflammatory process on the vascular endothelium of the skin during severe peritonitis. Cutaneous E-selectin expression thus reflected on the surface of the body a state of generalized activated endothelium.

Cell Adhesion Molecules↗
PubMed1990

Immunostaining of human brain capillaries by antibodies to very late antigens.

Normal and neurologically diseased brain tissue was stained with antibodies directed against very late antigens (VLAs) 1, 2, 3, 5, 6, their common beta 1-subunit, and three extracellular proteins: collagen type IV, laminin and fibronectin. Strong staining of capillaries was obtained in both normal and pathological tissue with antibodies to collagen type IV and its putative receptor VLA-1; laminin and its putative receptor VLA-6; and the common VLA beta 1-subunit. Only residual plasma in vessels was stained with the antibody to fibronectin. Negative staining was obtained with antibodies to the fibronectin receptor VLA-5 and VLA-3. Trace staining was obtained with the antibody to VLA-2.

Alzheimer Disease↗
PubMed2006

Multiple gastrointestinal stromal tumors of the ileum and neurofibromatosis type 1.

Type 1 neurofibromatosis, also known as von Recklinghausen disease, is one of the most common genetic disorders. Gastrointestinal associations have been well described in these patients, but the true incidence of gastrointestinal tumors and the proportion of these becoming clinically significant are not known. The most common gastrointestinal tumors are stromal tumors, most of which are located in the stomach and jejunum. We discuss the case of a female patient with neurofibromatosis whose initial diagnosis was an ovarian mass. During surgery the diagnosis of an intestinal stromal tumor was made. Operative findings were a multilobulated tumor arising from the ileal wall 50 cm from the ileocecal valve. The tumor did not originate from the nervous myenteric plexus or muscular layer of the small bowel wall; it originated from within the stromal cells of the intestinal wall. Mitotic count showed 3 mitoses per 10 high-power fields. Immunohistochemical stains of the tumor showed positive staining for CD117 and CD34 and negative staining for S100, alpha-smooth muscle actin, and desmin. The intestinal myenteric plexus showed positive staining for chromegranin A and S100. The histologic characteristics of this patient's tumor are compatible with an undifferentiated stromal tumor of nonneural or nonmuscular origin.

Adult↗
PubMed1998

Special techniques in diagnostic electron microscopy.

The power of electron microscopy as a diagnostic tool can be amplified considerably by the application of ancillary preparative and analytic methods. Subcellular chemistry and structure can be examined by various forms of microprobe analysis and by special staining methods, including cytochemical, immunocytochemical, and negative staining. Qualitative ultrastructural examination can be augmented by morphometric analysis. Correlative microscopic survey methods can be used as a means of targeting ultrastructural investigations. This article provides an overview of the use of these special techniques in the diagnosis and classification of tumors and other selected pathologic processes.

Diagnosis, Differential↗
PubMed2006

Involvement of a putative [Fe-S]-cluster-binding protein in the biogenesis of quinohemoprotein amine dehydrogenase.

Quinohemoprotein amine dehydrogenase (QHNDH) of Paracoccus denitrificans contains a peptidyl quinone cofactor, cysteine tryptophylquinone, as well as intrapeptidyl thioether cross-links between Cys and Asp/Glu residues within the smallestgamma-subunit of the alphabetagamma heterotrimeric protein. A putative [Fe-S]-cluster-binding protein (ORF2 protein) encoded between the structural genes for the alpha- and gamma-subunits of QHNDH in the n-butylamine-utilizing operon likely belongs to a Radical SAM (S-Ado-Met) superfamily that includes many proteins involved in vitamin biosynthesis and enzyme activation. In this study the role of ORF2 protein in the biogenesis of QHNDH has been explored. Although the wild-type strain of Paracoccus denitrificans produced an active, mature enzyme upon induction with n-butylamine, a mutant strain in which the ORF2 gene had been mostly deleted, neither grew in the n-butylamine medium nor showed QHNDH activity. When the mutant strain was transformed with an expression plasmid for the ORF2 protein, n-butylamine-dependent bacterial growth and QHNDH activity were restored. Site-specific mutations in the putative [Fe-S]-cluster or SAM binding motifs in the ORF2 protein failed to support bacterial growth. The alpha- and beta-subunits were both detected in the periplasm of the mutant strain, whereas the gamma-subunit polypeptide was accumulated in the cytoplasm and stained negatively for redox-cycling quinone staining. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that the gamma-subunit isolated from the mutant strain had not undergone posttranslational modification. These results unequivocally show that the putative [Fe-S]-cluster- and SAM-binding ORF2 protein is necessary for the posttranslational processing of gamma-subunit, most likely participating in the formation of the intrapeptidyl thioether cross-links.

Amino Acid Sequence↗
PubMed1988

Fibrous papules. A light microscopic and immunohistochemical study.

Thirty-six cases of fibrous papules of the face and nose were studied by light microscopy and by immunohistochemistry. Positive staining with antibodies to vimentin and negative staining with antibodies to S-100 protein, alpha 1-antichymotrypsin, and factor VIII-RAG of the spindle, stellate, and multinucleated cells observed in the dermis support a fibroblastic line of differentiation. Fibrous papules of the nose and face are a distinctive clinicopathologic entity that most probably represents an inflammatory rather than a neoplastic process, sharing some histologic features of angiofibromas (angiofibrosis) and perifollicular fibromas (perifollicular fibrosis).

Adult↗
PubMed1990

Distribution of the protease inhibitor alpha 1-antichymotrypsin in cerebral and systemic amyloid.

We performed immunocytochemical staining to study the distribution of serum protease inhibitors in cerebral and systemic amyloid deposits. In beta-protein amyloid deposits in Alzheimer's disease, Down's syndrome, age-related cerebral amyloidosis, sporadic cerebral amyloid angiopathy and hereditary cerebral hemorrhage with amyloidosis of Dutch origin, antibody to alpha 1-antichymotrypsin (ACT) stains senile plaques and vascular deposits. Immature plaques or preamyloid deposits, identified by their positive staining for beta-protein and negative staining for Congo red, which represents the earliest recognizable stages of amyloid deposition, are also labeled. We did not detect ACT in other chemically different forms of cerebral and systemic amyloid. None of the other inhibitors in this study, i.e. antithrombin III and alpha 2-macroglobulin, was detected in the amyloid deposits. Neurons and glial cells throughout the central nervous system in normal and amyloid-containing brains also bind ACT antibody. The results emphasize the close association of ACT with one type of cerebral amyloid (beta-amyloid diseases) as well as the failure to detect such an association in other chemically different forms of cerebral and systemic amyloids.

Amyloid↗
PubMed1977

Preparation of thin cryo-sections for electron probe analysis of calcifying cartilage.

Conventional methods of fixation, dehydration, embedding and wet-sectioning can produce artefacts in the chemical composition of mineralizing tissues. Cryoultramicrotomy was adopted for a more reliable approach to electron probe analysis of initial apatite formation in calcifying cartilage. Fresh rabbit epiphyseal cartilage was mounted on silver pins, frozen by immersion in liquid nitrogen, and sectioned with the specimen temperature at 133 K and the knife temperature at 273 K. Dry cryo-sections (30-70 nm in thickness) were manipulated on to coated grids and examined the same day. These cryo-sections showed good morphological and cytoplasmic detail, with large areas relatively free of ice-crystal damage. Sections stained either with osmium vapour or negatively stained with silicotungstic acid showed areas with well-preserved mitochondria with granules and endoplasmic reticulum. Unstained sections also showed dense granules (50-120 nm in diameter) in the mitochondria of chondrocytes and preliminary electron probe analysis of these has indicated a Ca/P mass ratio of approximately 1.14. In the longitudinal septa, about 2 micrometer away from the chondrocytes, matrix-vesicle-like particles could be seen with crystal needles inside them. Micro-analysis of two of these gave a Ca/P mass ratio of 1.73 and 2.68. Cryo-ultramicrotomy appears to confirm a number of conclusions derived from conventional ultrastructural study of growth cartilage and suggests for the first time how amorphous calcium phosphate and crystalline apatite can be shown to exist in different organelles in the same cryo-section of the tissue.

Animals↗
PubMed2005

Intestinal-type mucinous adenocarcinoma arising from the prostatic duct.

We present a case of mucinous adenocarcinoma of intestinal type arising from the prostatic duct in a 72-year-old Japanese man. The patient presented with macroscopic hematuria. Cystourethroscopy exhibited a mucus deposit at the 5 o'clock position of the verumontanum portion. A transurethral biopsy specimen revealed mucinous adenocarcinoma. A radical retropubic prostatectomy was performed. In the prostatectomy specimen, the cancer lesion mainly showed intraductal growth in the prostatic ducts with scattered mucin lakes in the prostatic stroma. There were no abnormalities in the urethral epithelium. The cancer cells resembled the intestinal epithelium rather than either the prostatic duct or the acinar epithelium, which showed diffusely positive immunohistochemical staining for carcinoembryonic antigen, but showed negative staining for prostate-specific antigen. Therefore, these findings suggest mucinous adenocarcinoma of intestinal type arising from the prostatic duct. A number of cases with mucinous adenocarcinoma arising from the prostatic urethra resembling the present case have been reported, but this is the first known case of carcinoma arising from the prostatic duct.

Adenocarcinoma, Mucinous↗
PubMed2001

Tumour infiltrating lymphocytes and apoptosis are independent features in colorectal cancer stratified according to microsatellite instability status.

BACKGROUND: The presence of high level DNA microsatellite instability (MSI-H) in colorectal cancer is associated with an improved prognosis, as is the presence of tumour infiltrating lymphocytes (TILs). It is not clear if TILs contribute directly to the survival advantage associated with MSI-H cancers through activation of an antitumour immune response. AIMS: To correlate TIL and apoptosis rates in colorectal cancer stratified by MSI status. METHODS: The distribution of TILs was characterised and quantified in a selected series of 102 sporadic colorectal cancers classified according to levels of MSI as 32 MSI-H, 30 MSI-low (MSI-L), and 40 microsatellite stable (MSS). Archival blocks were immunostained using the T cell markers CD3 and CD8, and the B cell marker CD20. Apoptosis of malignant epithelial cells was quantified by immunohistochemistry with the M30 CytoDEATH antibody. RESULTS: Positive staining with anti-CD3 and negative staining with anti-CD20 identified virtually all TILs as T cells. The majority of CD3+ TILs (>75%) also stained with anti-CD8. TILs were most abundant in MSI-H colorectal cancers in which 23/32 (72%) scored as TIL positive. Only 5/40 (12.5%) MSS tumours and 9/30 (30%) MSI-L cancers were TIL positive (p<0.0001). MSI-H cancers showed a twofold higher rate of apoptosis (mean (SD) 3.52 (0.34)%) than the MSS cancers (1.53 (0.23)%) while the MSI-L subgroup had an intermediate level (2.52 (0.35)%) (p<0.0001). Overall, there was a small (r=0.347) but significant linear correlation between CD3+ and M30+ random apoptosis counts (p<0.001). However, TILs and apoptosis showed little colocalisation. CONCLUSIONS: While TILs might be expected to explain the increased apoptotic rate and improved prognosis of MSI-H cancers, it is likely that TILs and apoptosis are independent characteristics of MSI-H cancers.

Adenocarcinoma↗