Endothelin-1 genetic polymorphism as predictive marker for bevacizumab in metastatic breast cancer.

Biomarkers for bevacizumab efficacy in metastatic breast cancer (MBC) are of urgent need. The genetic variability of genes involved in angiogenesis could explain the interpatient variability of drug effects. For this biomarker study DNA was extracted from tumor blocks or blood samples of patients with human epidermal growth factor receptor 2 (HER2)-negative MBC treated with bevacizumab in combination with chemotherapy (bevacizumab cohort, 163 patients) or chemotherapy only (control cohort, 105 patients). We assessed the correlation of 10 single-nucleotide polymorphisms (SNPs) in genes modulating angiogenesis (vascular endothelial growth factor-A (VEGF-A), VEGF receptor 1 (VEGFR-1), serine threonine kinase 39 (STK39)) or hypertension (endothelin-1 and uromodulin) with outcome and toxicity. In the bevacizumab cohort, the SNP rs5370-TT in endothelin-1 (EDN1) showed a significantly shorter median overall survival (OS, 6.3 vs 21.3 months; hazard ratio (HR) 2.89, 95% confidence interval (CI) 1.34-6.26; log-rank P=0.0069) and a trend toward worse median progression-free survival (3.5 vs 7.9 months; HR 2.05, 95% CI 0.96-4.39; log-rank P=0.065) compared with the alternate genotypes combined. Similarly, patients harboring the VEGF-936 (rs3025039) TT alleles showed a significantly shorter median OS than patients with VEGF-936 CC or CT (14.9 vs 21.3 months; HR 2.37, 95% CI 1.09-5.13; P=0.0286). In multivariate analysis including important clinical parameters like disease-free survival (DFS), adjuvant chemotherapy, ECOG (Eastern Cooperative Oncology Group) performance score, histologic subtype, grade, hormone receptor status, visceral metastases and treatment line, only the association of rs5370 (EDN1) with OS was still statistically significant (P=0.012). In the control cohort, no association of the EDN1 genotype with outcome was seen, suggesting a predictive value for bevacizumab. In conclusion, the SNP rs5370 in endothelin-1 could help identifying patients who unlikely gain any benefit from bevacizumab.

Transcutaneous immunotherapy via laser-generated micropores efficiently alleviates allergic asthma in Phl p 5-sensitized mice.

BACKGROUND: Specific immunotherapy via the subcutaneous or oral route is associated with local and, in some cases, systemic side effects and suffers from low patient compliance. Due to its unique immunological features, the skin represents a promising target tissue for effective and painless treatment of type I allergy. The current study was performed to compare the efficacy of transcutaneous immunotherapy via laser-generated micropores to subcutaneous injection. METHODS: BALB/c mice were sensitized by intraperitoneal injection of recombinant grass pollen allergen Phl p 5 together with alum. Subsequently, lung inflammation was induced by repeated intranasal challenge. During the treatment phase, adjuvant-free Phl p 5 was applied in solution to microporated skin or was subcutaneously injected. Lung function and cellular infiltration; Phl p 5-specific serum levels of IgG1, IgG2a, and IgE; and cytokine levels in bronchoalveolar lavage fluids as well as in supernatants of splenocyte cultures were assessed. RESULTS: Both therapeutic approaches reduced airway hyperresponsiveness and leukocyte infiltration into the lungs. Whereas subcutaneous immunotherapy induced a systemic increase in Th2-associated cytokine secretion, transcutaneous application revealed a general downregulation of Th1/Th2/Th17 responses. Successful therapy was associated with induction of IgG2a and an increase in FOXP3+ CD4+ T cells. CONCLUSIONS: Transcutaneous immunotherapy via laser microporation is equally efficient compared with conventional subcutaneous treatment but avoids therapy-associated boosting of systemic Th2 immunity. Immunotherapy via laser-microporated skin combines a painless application route with the high efficacy known from subcutaneous injections and therefore represents a promising alternative to established forms of immunotherapy.

Cooperation between GLI and JUN enhances transcription of JUN and selected GLI target genes.

Sustained Hedgehog (HH) signaling is implicated in basal cell carcinoma of the skin and other types of cancer. Here we show that GLI1 and GLI2, the main transcriptional activators of the HH pathway, directly regulate expression of the activator protein 1 (AP-1) family member JUN, a transcription factor controlling keratinocyte proliferation and skin homeostasis. Activation of the JUN promoter by GLI is dependent on a GLI-binding site and the AP-1 sites known to be involved in self-activation of JUN. Transcription of JUN is greatly enhanced in the presence of GLI and requires activated JUN protein. GLI2act is a more potent activator than GLI1 in these experiments and physical interaction with phosphorylated JUN was only detected for GLI2act. The synergistic effect of GLI and JUN extends to the activation of further GLI target genes as shown by shRNA-mediated knockdown of JUN in human keratinocytes. Some of these cooperatively activated genes are involved in cell-cycle progression, which is consistent with a significant reduction of the proliferative potential of GLI in the absence of JUN. These results suggest a novel connection between HH/GLI pathway activity and JUN, which may contribute to the oncogenic activity of HH/GLI signaling in skin.

Novel approach of human epidermal growth factor receptor 2 detection in noninvasive and invasive transitional cell carcinoma of the bladder.

PURPOSE: In recent years over expression of HER2 has been identified in malignant tumors of organs other than breast. Studies of bladder carcinoma that analyzed HER2 protein expression and gene amplification with a variety of nonstandardized methods have shown heterogeneous results. The results reported vary from 2% to 74% of protein over expression, to 4% to 59% of gene amplification of HER2, possibly due to differences in study design, material selection or laboratory methodology. MATERIALS AND METHODS: In the present study archival tissue from 87 patients with noninvasive papillary (25) and invasive (62) TCC was analyzed for amplification of the HER2 gene and over expression of its encoded protein. HER2 protein expression was detected by immunohistochemistry using the HercepTest. Routinely processed paraffin embedded tissue was investigated for HER2 gene amplification using CISH and FISH. RESULTS: Of the invasive 37 (58%) and of the noninvasive 19 (76%) transitional cell carcinomas investigated showed over expression of the HER2 protein (3+ and 2+) using a standardized immunohistochemical method. HER2 gene amplification assays performed on positive cases evaluated by immunohistochemistry were obtained in 81% and 43% of 3+ and 2+ HER2 protein over expressing invasive, respectively, and in 21% of noninvasive papillary bladder tumors. HER2 gene amplification detection results using CISH and FISH showed a concordance of 100%. The occurrence of aneusomy of chromosome 17 in association with HER2 gene amplification was investigated. CONCLUSIONS: Validation of the HER2 oncogene in bladder cancer may allow for the potential use of Herceptin(R) antibody therapy. Therefore, the appropriate treatment approach has to be based on reliable and standardized analysis. Our results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 oncogene amplification in bladder cancer.

Angiogenic potential of ductal carcinoma in situ (DCIS) of human breast.

AIMS: Comedo carcinoma is generally regarded as the subtype of ductal carcinoma in situ (DCIS) most likely to progress to invasive carcinoma. Increased angiogenesis could be associated with an enhanced risk of progression and might therefore be a marker of poor prognosis, as can be demonstrated for invasive breast tumours. Therefore, the present study investigates the correlations between the expression of oncoproteins (HER2, HER1/EGFR), angiogenic growth factors (VEGF and PD-ECGF/TP) and microvessel density (MVD) in DCIS. METHODS AND RESULTS: Forty-six breast cancer specimens of DCIS were tested immunohistochemically for the expression of angiogenic factors and oncoproteins. Different vascular distribution patterns of DCIS were examined semiquantitatively. Our results showed a significantly inverse correlation between HER1/EGFR and comedo-type DCIS (P = 0.048), but HER1/EGFR expression seemed to be independent of HER2 overexpression. VEGF expression was significantly associated with endoglin expression (P = 0.031) and the cuffing phenomenon (P = 0.017). CONCLUSIONS: The significantly inverse correlation between HER1/EGFR and comedo-type DCIS and the observation that VEGF and the other angiogenic factors tested are independent of HER2 overexpression, suggest that progression of comedo-type DCIS and angiogenesis in breast carcinoma are not regulated via the HER1/EGFR or HER2 pathway.

Activation of the MAP kinase pathway induces chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) expression in human breast cancer cell lines.

Growth factors are essential for cellular growth and differentiation in both normal and malignant human breast epithelial cells. In the present study we investigated the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGFalpha) and phorbol myristate acetate (PMA) on chicken ovalbumin upstream promoter-transcription factor (COUP-TF) expression in human breast cancer cells. The orphan receptors COUP-TFI and COUP-TFII are members of the nuclear receptor superfamily. The high degree of evolutionary conservation of these proteins strongly argues for an important biological function. COUP-TF expression was highest in SK-BR3 cells (approximately 130 amol/ micro g total RNA), while the lowest COUP-TF expression was observed in MCF-7 cells (3.5 amol/ micro g total RNA). While treatment of EGF, TGFalpha and PMA induced expression of COUP-TFII, COUP-TFI did not respond to these agents. Oncostatin M (OSM) is known to exert an antiproliferative effect in breast cancer cells. Treatment of MCF-7 cells with OSM resulted in an approximately 90% reduction of COUP-TFII mRNA expression. In SK-BR3 cells, treatment with the MEK inhibitor UO126 resulted in a profound suppression of endogenous COUP-TFII expression. Furthermore, cotreatment with UO126 prevented induction of COUP-TFII expression by EGF in MCF-7 cells. In conclusion, our data provide evidence, for the first time, that mitogenic substances which activate the MAP kinase pathway, can induce COUP-TFII expression. Our results strongly suggest that an active MAP kinase pathway is essential for COUP-TFII expression in human breast cancer cells.

Immunohistochemical assessment of an asymptomatic glucagonoma in a patient with hypergastrinemia and marked antral angiodysplasia.

A 58-year-old patient had been treated for recurrent gastritis. Numerous gastroscopies indicated hemorrhagic gastritis combined with increasingly severe anemia. The patient was admitted with a hemoglobin of 4.4 g/dL. Gastroscopy showed marked antral angiodysplasia. Serum samples for gastrin were taken and found to be elevated (170-250 U/mL). The search for a gastrin-producing tumor with abdominal ultrasound, computed tomography, octreotide scan, and secretin test was negative, but angiography detected a pancreas tumor with a 2-cm diameter. Partial pancreatectomy and partial gastrectomy were performed. Immunohistochemical examination of the tumor did not show a gastrinoma but did show glucagon-reactive tissue. Further tumors or elevated plasma hormone levels were not detected, and a multiple endocrine neoplasia type I syndrome could be excluded. We thus found antral angiodysplasia with hypergastrinemia leading to detection of a glucagonoma diagnosed by immunohistochemistry. After more than 4 years of follow-up, the patient is without any symptoms or signs of relapse or secondary hormone syndrome.

Co-expression of tenascin-C and vimentin in human breast cancer cells indicates phenotypic transdifferentiation during tumour progression: correlation with histopathological parameters, hormone receptors, and oncoproteins.

Loss of epithelial morphology and the acquisition of mesenchymal characteristics are typical for carcinoma cells in tumour progression. In human breast carcinomas, up-regulation of tenascin-C (TN-C) and vimentin (Vim) is frequently observed in cancer cells and correlates with increased malignancy. Thus, it is possible that TN-C is co-expressed with Vim, representing cancer cells that have undergone epithelial-mesenchymal transition (EMT). This study examined 128 breast carcinomas using immunohistochemical techniques to demonstrate that mammary cancer cells are a prominent source of both TN-C and Vim. Statistical analysis revealed a significant association between TN-C and Vim expression in cancer cells. TN-C expression also correlated positively with overexpression of c-erbB-2 oncoprotein and down-regulation of oestrogen receptors (ERs). Eleven human mammary cancer cell lines and two 'normal' cell lines were examined by western blotting and immunohistochemistry. Co-expression of TN-C and Vim was detected in the carcinosarcoma cell line HS 578T, SK-BR-3 (B), fibroblast-like MDA-MB-231 cells, and the myoepithelial cell line HBL 100. These findings suggest that TN-C and Vim, when co-expressed in mammary carcinoma cells, represent regulator genes likely to be involved in EMT during mammary carcinogenesis.

Detection of human papillomavirus in cervical carcinoma: comparison of peroxidase, Nanogold, and catalyzed reporter deposition (CARD)-Nanogold in situ hybridization.

We compared three in situ hybridization (ISH) methods for their applicability and sensitivity in detecting human papillomavirus (HPV) in 61 cases (1 Grade 1, 18 Grade 2, 42 Grade 3) of routinely processed squamous cell cervical carcinoma. A commercially available biotinylated probe for HPV-16/18 was applied to serial sections and detected by conventional streptavidin-biotin-peroxidase ISH, streptavidin-Nanogold-silver ISH, and catalyzed reporter deposition (CARD)-Nanogold-gold ISH. The latter method involved signal amplification by peroxidase-catalyzed deposition of biotinylated tyramides at the hybridization sites, followed by detection of accumulated biotin by streptavidin-Nanogold made visible by autometallography. The HPV-16/18 detection rates for the three methods were 39.3, 44.3, and 65.6%, respectively. In all of the three ISH methods, a punctate staining pattern (single or multiple intranuclear spots of variable size), presumably indicating viral integration, was highly predominant among the positive cases. Two of the cases identified as positive by streptavidin-biotinperoxidase ISH were rated negative with streptavidin-Nanogold-silver ISH, whereas six cases that were clearly negative with streptavidin-biotinperoxidase ISH became positively stained with streptavidin-Nanogold ISH. All of these discordant cases were positive by the highly sensitive CARD-Nanogold-gold ISH. In addition, the high detection sensitivity of CARD-Nanogold-gold ISH was confirmed by its ability to detect single copies of HPV-16 in SiHa cells. In general, we found that the intense black reaction product from Nanogold autometallography gave superior contrast to that obtained with the peroxidase system. After tyramide signal amplification, the staining was so clearly visible that preparations could be readily screened under low magnification. Our findings precisely demonstrated the need for improved sensitivity in the in situ detection of HPV. The CARD-Nanogold-gold technology looks promising as a highly sensitive method for routine ISH in molecular pathology.

Peptidergic innervation of the human clitoris.

In the present study, the distributions of neuropeptides in the normal human clitoris and in a clitoris from an adrenogenital syndrome (AGS) was demonstrated by immunohistochemistry (IHC). Immunohistochemical screening detected a complex network of nerve fibers containing vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), neuropeptide tyrosine (neuropeptide Y), C-flanking peptide of neuropeptide Y (CPON), calcitonin gene-related peptide (CGRP) and substance P immunoreactivities. Special attention was given to the VIP-related peptide helospectin, that has been detected in neuronal elements in the clitoris. No visible differences between the localization and distribution of peptidergic nerve fibers of normal and hypertrophic clitoris from AGS have been observed. Co-localization studies showed the co-existence of VIP, PHM and partly helospectin and neuropeptide Y with CPON within nerve fibers in the cavernous tissue and substance P and CGRP co-expression in nerve fibers especially underneath and within the glans clitoris.

p34cdc2 in invasive breast cancer: relationship to DNA content, Ki67 index and c-erbB-2 expression.

AIMS: One-hundred and eighty-eight cases of human mammary carcinoma were examined immunohistochemically for their expression of Ki67, p34cdc2 and c-erbB-2. DNA image cytometry was performed to evaluate DNA ploidy, Auer type, S-phase fraction (SPF), 5c exceeding rate (5cER) and 2c deviation index (2cDI). METHODS AND RESULTS: One-hundred and sixty-eight cases were invasive ductal carcinomas, 20 were of invasive lobular type. Routinely assessed oestrogen and progesterone receptor scores were available. The results were analysed statistically in comparison to tumour type, histopathological grade, lymph node status, menopausal status, patient age and overall survival. Ki67 (P < 0.002) and c-erbB-2 (P < 0.0001) correlated well with overall survival (P < 0.0008) and grade (P < 0.038) but not with lymph node status and tumour type. p34cdc2 showed a trend towards a positive correlation with Ki67 (P < 0.058) and a significant negative correlation with receptor status (P < 0.008) but with none of the other parameters examined. CONCLUSIONS: No association between the DNA measured parameters (Auer type, SPF, 5cER and 2cDI) and survival was found. Our results suggest that c-erbB-2 and Ki67 are parameters which might, in combination with receptor status, help to define subgroups with different outcomes.

Nitric oxide synthase in the innervation of the human nasal mucosa: correlation with neuropeptides and tyrosine hydroxylase.

Nitric oxide (NO) is a powerful mediator in the central and peripheral nervous system. In the present study the authors have examined the human nasal mucosa innervation for the presence of the neuronal isoform of the NO-generating enzyme, NO-synthase (NOS), and its correlation with other neuronal mediators and markers by means of double-labeling immunohistochemistry. NOS-immunoreactive nerve fibers were observed to be numerously present around glands and venous sinusoids and, less frequently, around small arteries and veins. Few fibers were seen in the lamina propria. NOS appeared to be frequently colocalized in nerve fibers with vasoactive intestinal peptide and, occasionally, with substance P and tyrosine hydroxylase, a marker for catecholamine biosynthesis. These findings suggest that neurally released NO is an important regulatory mediator of glandular secretion and blood flow in the nasal mucosa.

Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma.

Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic studies addressing ISH fixation conditions have been published. We reasoned that heavy metals present in some precipitating fixatives may compromise duplex formation during ISH. Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) and two negative cell lines (K562 and MOLT 4) were expanded to >10(10) and pellets fixed in NBF, zinc formalin, B5, and Bouin's and Hollande's solutions, and subjected to DNA ISH using biotinylated genomic probes. Ten tissue biopsies fixed in both Hollande's and NBF solutions were also evaluated for human papilloma virus content using DNA ISH. Additionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with fluorescein isothiocyanate-labeled oligonucleotides. Catalyzed reporter deposition combined with Streptavidin-Nanogold staining and silver acetate autometallography (Catalyzed reporter deposition-Ng-autometallography ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals entrapped in fixed tissues were removed by two exposures to Lugol's iodine. Results for both DNA and RNA ISH comparing B5 and NBF fixatives were virtually identical. Hollande's, Bouin's, B5, and zinc formalin fixed tissue showed results indistinguishable from NBF fixed tissue in DNA ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions.

Localization of endothelin-1 and endothelin-3 in the cochlea.

The distribution of endothelin-1 (ET-1) and endothelin-3 (ET-3) was studied by indirect immunostaining of decalcified guinea pig and rat cochleae. No species differences were observed. Perikarya and processes of spiral ganglion cells were highly reactive for both ET-1 and ET-3. The epithelial lining of the cochlear duct stained for ET-1 and ET-3, but reactivity for ET-1 was higher in the lining cells of the inner sulcus, Claudius', and Hensen's cells, while the tympanic covering layer of the basilar membrane stained stronger for ET-3 compared to ET-1. In the stria vascularis, all cell types stained for ET-3, while marginal cells were more reactive for ET-1. Spiral ligament fibroblasts were reactive for ET-1, but not for ET-3. Connective tissue cells of the spiral limbus stained for both endothelins. The region of synapses on outer hair cells reacted for ET-1 and ET-3 but sensory cells remained unstained. Endothelins are discussed to act as modulatory peptides, possibly interfering with nitric oxide, prostaglandins, and atrial natriuretic peptide in the lateral cochlear wall (lateral cochlear wall, i.e. stria vascularis and spiral ligament). The occurrence of endothelins in cochlear neurons suggest their potential role as neurotransmitters.

Sensitive in situ hybridization with catalyzed reporter deposition, streptavidin-Nanogold, and silver acetate autometallography: detection of single-copy human papillomavirus.

The usefulness of standard in situ hybridization for viral nucleic acid detection is occasionally limited by its sensitivity limit of 10 to 50 copies per cell. A modified version of the recently described signal amplification method, catalyzed reporter deposition (CARD), and its application to formalin-fixed cells and tissue sections is presented. Deposition of the reporter is facilitated by using horseradish peroxidase catalyzing the deposition of biotinylated tyramide on the location of the probe target. The biotin accumulation created is usually detected with streptavidin-labeled enzymes or fluorochromes. In the present investigation, this step was replaced by streptavidin-Nanogold and combined with silver acetate autometallography. This resulted in deep-black precipitation at positive in situ hybridized reaction sites. The sensitivity of this new approach was tested with a biotinylated, genomic probe specific for human papillomavirus (HPV)-16/18. SiHa cells, a cervical carcinoma-derived cell line with one to two HPV16 copies per cell, and 10 histologically confirmed cervical carcinomas were used for the study. All samples were previously HPV16 positive with solution polymerase chain reaction, but only two of the cervical carcinomas were positive with standard in situ hybridization with barely visible signals. When employing CARD-Nanogold, SiHa cells and 9 of 10 biopsies proved positive with marked signals. It is concluded that this nonisotopic method can detect single viral copies in situ in routinely fixed material and may have the potential to replace in situ polymerase chain reaction in many applications.

CGRP and substance P in intraepithelial neuronal structures of the human upper respiratory system.

The distribution of intraepithelial nerve fibres and neuroendocrine cells within the surface and glandular epithelium of human nasal mucosa and larynx was examined using immunohistochemical techniques. Neuronal structures were immunostained for the general neuroendocrine marker protein gene-product (PGP) 9.5, and the two neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) using immunofluorescence and streptavidin-biotin-peroxidase complex (S-ABC) methods. Intraepithelial nerve fibres with free nerve endings contained PGP 9.5 and were found within the respiratory surface epithelium of the nasal mucosa and the squamous epithelium of the larynx. A subpopulation of these nerve fibres showed positive immunoreactivties with antibodies against SP and CGRP. Nerve fibres within the ductal epithelium of subepithelial excretory ducts passing the basal membrane and reaching the luminal part were detected. These nerve fibres showed CGRP-like immunoreactivity but not for SP. A dense network of nerve fibres within the squamous surface epithelium was detected in the subglottic and epiglottic region containing CGRP and SP in a small subpopulation of nerve fibres. Single intraepithelial taste buds in the epiglottic region and neuroendocrine cells within the subglottic epithelium expressed PGP 9.5.

Electron Microscopical Autometallography: Immunogold-Silver Staining (IGSS) and Heavy-Metal Histochemistry

Immunogold-silver staining (IGSS) utilizes a histochemical method called autometallography (AMG) to amplify tiny gold particles to sizes easily visible both in light and electron microscopy. In both applications it is advisable to use the smallest possible gold diameters (1-6 nm) to obtain the highest sensitivity, thus, allowing minute amounts of the target substance to be demonstrated. Gold labels smaller than 10 nm in diameter have been clearly shown to give the highest labeling densities of antigen-antibody binding sites. AMG can be used for the detection of catalytic crystal lattices of metallic gold and silver, and sulfides or selenides of mercury, silver, copper, bismuth, and zinc. The method has its roots in "physical development" technique, transplanted from photography to histology by Liesegang at the beginning of this century. In 1981, a series of papers were published by one of us with the purpose of introducing a reliable and easy-to-handle technique for light microscopical and ultrastructural studies. AMG has a multitude of applications apart from its use in detecting tissue metals. These include the highly sensitive and efficient in situ colloidal gold tracing of peptides, proteins, and amines by immunocytochemistry using the IGSS method, of carbohydrates by lectin IGSS, and of nucleic acids by IGSS in situ hybridization, IGSS in situ polymerase chain reaction, and IGSS in situ self-sustained sequence replication-based amplification (in situ 3SR) techniques, the last two even performing with single-copy sensitivity. Applications of pre- and postembedding AMG for semithin and ultrathin tissue sections are described.