Impact of oncogenic BRAF mutations and p16 expression on the growth rate of early melanomas and naevi in vivo.

BACKGROUND: It is important to know what drives and arrests melanocytic growth in vivo but observations linking oncogenic mutations to growth rates of melanocytic neoplasms in vivo are sparse. OBJECTIVES: To clarify the relationship between BRAF(V) (600E) mutations and p16 expression and the growth rate of melanocytic neoplasms in vivo. METHODS: We measured the growth rate of 54 melanocytic lesions (26 melanomas, 28 naevi) in vivo with digital dermatoscopy and correlated it with BRAF(V) (600E) and p16 expression, and with dermatoscopic and histological patterns. RESULTS: Melanomas grew faster than naevi (mean 2·7 vs. 0·8 mm(2) /year; P < 0·001) and the growth rate was faster in lesions with more nests (> 25% nests: 2·0 mm(2) /year vs. < 25% nests: 1·0 mm(2) /year; P = 0·036). Melanomas with the BRAF(V) (600E) mutation grew significantly faster than melanomas without the mutation (mean 3·36 vs. 1·60 mm(2) /year, P = 0·018). This effect of the BRAF(V) (600E) mutation on the growth rate was not observed in melanocytic naevi (mean 1·01 vs. 0·47 mm(2) /year, P = 0·274). Histopathologically, extensive nesting, larger nests and larger cell sizes were more common in melanocytic neoplasms with the BRAF(V) (600E) mutation than in those without the mutation. Melanomas expressing p16 had a slower growth rate than melanomas without p16 expression (2·27 vs. 4·34 mm(2) /year, P = 0·047). This effect was not observed in naevi (0·81 vs. 0·68 mm(2) /year, P = 0·836). CONCLUSIONS: The expression of BRAF(V) (600E) and the loss of p16 accelerate the growth rate of early melanomas in vivo but not in melanocytic naevi. In comparison to melanocytic proliferations that lack the mutation, the epidermal melanocytes in lesions that harbour BRAF(V) (600E) mutations are larger and more frequently arranged in large nests.

RCAS-1 serum and tumor levels in head and neck squamous cell carcinoma.

BACKGROUND/AIMS: RCAS-1 is a transmembrane protein that is involved in the evasion of host immune surveillance by tumor cells. It has been found to be a valuable prognostic and diagnostic marker in a number of different malignancies. The objective of the study was to analyze the potency of RCAS-1 as a biomarker in the serum of patients with head and neck squamous cell carcinoma (HNSCC). METHODS: ELISA was performed with prospectively collected serum samples from 60 patients with HNSCC (taken at the time of diagnosis, after 3 and 12 months) and from 31 healthy controls. To correlate serum levels with RCAS-1 expression in the tumor, immunohistochemical staining of RCAS-1 was done using a tissue microarray. RESULTS: Surprisingly, median sRCAS-1 levels were basically identical between tumor patients and controls. Interestingly, patients with low RCAS-1 values at the time of diagnosis had better disease-free survival. 62% of tumor samples expressed RCAS-1 but we could not demonstrate a correlation between protein expression and serum levels. CONCLUSION: This study was the first to correlate RCAS-1 levels in the serum and in the tumor of the same patients. RCAS-1 seems to have prognostic properties although larger studies will be necessary to fully evaluate its role in HNSCC.

Psoriasin (S100A7) is a major Escherichia coli-cidal factor of the female genital tract.

The female urogenital tract requires an efficient defense against bacteria, potentially derived from the adjacent intestinal tract. We have thus sought to identify the factors that protect against Escherichia coli (E. coli) in the female genital tract. Vaginal fluid from healthy human donors consistently killed E. coli in vitro and vaginal epithelium strongly expressed and secreted psoriasin. Psoriasin was constitutively produced in an organotypic vaginal epithelium model, and exposure of these cells to supernatants of E. coli cultures led to an enhanced psoriasin expression. Secreted psoriasin in vaginal fluids accounted for approximately 2.5-3% of total protein. Fractionation of vaginal fluids by high performance liquid chromatography (HPLC) showed that psoriasin co-eluted with a peak of E. coli killing activity. Our data show that normal vaginal fluid contains a powerful intrinsic antimicrobial defense against E. coli and that psoriasin contributes to the innate immune response of the female genital tract.

Strong evidence for up-regulation of cyclooxygenase-1 in head and neck cancer.

BACKGROUND: Cyclooxygenase-1, in contrast to cyclooxygenase-2, is generally reported to be constitutively expressed as a housekeeping enzyme in many different tissues. A number of recently published reports, however, challenge the notion that cyclooxygenase-1 expression is invariably constitutive by demonstrating that this enzyme might be up-regulated under certain pathological conditions in, for example, breast or ovarian cancer cells. In this study we investigated the expression of cyclooxygenase-1 in head and neck tumours and compared it to the cyclooxygenase-1 expression levels in normal oropharyngeal epithelial cells. MATERIAL AND METHODS: Paraffin-embedded pretreatment biopsies were obtained from head and neck tumour patients (n = 35). In addition, samples of normal oropharyngeal mucosa were taken from patients (n = 12) undergoing routine tonsillectomy. Cyclooxygenase-1 expression levels were determined by immunohistochemistry, Western blotting and real-time RT-PCR in cancerous tissue versus normal mucosa. RESULTS: Immunohistochemistry revealed overexpression of cyclooxygenase-1 in tumour biopsies compared to normal mucosa. Cyclooxygenase-1 was highly synthesized in the cytoplasm of neoplastic cells while significantly lower levels were detectable in normal mucosal cells. Western blotting and real-time RT-PCR also demonstrated higher cyclooxygenase-1 levels in tumour specimens compared to normal tissue samples. CONCLUSION: In this study we show for the first time that cyclooxygenase-1 is overexpressed in head and neck cancer cells compared to epithelial cells of normal mucosa.

TuBaFrost: European virtual tumor tissue banking.

TuBaFrost is a consortium responsible for the task to create a virtual European human frozen tumor tissue bank, composed of high quality frozen tumor tissue collections with corresponding accurate diagnosis stored in European cancer centers and universities, searchable on the Internet, providing rules for access and use and a code of conduct to comply with the various legal and ethical regulations in European countries. Such infrastructure would enlarge tissue availability and accessibility in large amounts of specified or even rare tumor samples. Design of an infrastructure for European residual tissue banking with the described characteristics, clear focus points emerge that can be broken down in dedicated subjects: (1) standardization and quality assurance (QA) to avoid inter-institute quality variation; (2) law and ethics enabling exchange of tissue samples possible between institutes in the different European countries, where law and ethics are characterized by a strong variability; (3) rules for access, with sufficient incentives for collectors; (4) central database application containing innovations on search and selection procedures; (5) support when needed with histology images; and (6) Internet access to search and upload, with in addition a solid website giving proper information on the procedures, intentions and activities not only to the scientific community, but also to the general public. One consortium decision, part of the incentives for collectors, had major impact on the infrastructure; custodianship over the tissues as well as the tissues stay with the collector institute. Resulting in specimens that are not given to an organization, taking decisions on participation of requests, but instead the local collected tissues stay very easy to access by the collector and allows autonomous negotiation between collector and requestor on cooperation, coauthorship in publication or compensation in costs. Thereby, improving availability of large amounts of high quality samples of a highly specified or rare tumor types and contact opportunities for cooperation with other institutes.

Virtual microscopy in virtual tumor banking.

Many systems have already been designed and successfully used for sharing histology images over large distances, without transfer of the original glass slides. Rapid evolution was seen when digital images could be transferred over the Internet. Nowadays, sophisticated virtual microscope systems can be acquired, with the capability to quickly scan large batches of glass slides at high magnification and compress and store the large images on disc, which subsequently can be consulted through the Internet. The images are stored on an image server, which can give simple, easy to transfer pictures to the user specifying a certain magnification on any position in the scan. This offers new opportunities in histology review, overcoming the necessity of the dynamic telepathology systems to have compatible software systems and microscopes and in addition, an adequate connection of sufficient bandwidth. Consulting the images now only requires an Internet connection and a computer with a high quality monitor. A system of complete pathology review supporting biorepositories is described, based on the implementation of this technique in the European Human Frozen Tumor Tissue Bank (TuBaFrost).

TuBaFrost 6: virtual microscopy in virtual tumour banking.

Many systems have already been designed and successfully used for sharing histology images over large distances, without transfer of the original glass slides. Rapid evolution was seen when digital images could be transferred over the Internet. Nowadays, sophisticated Virtual Microscope systems can be acquired, with the capability to quickly scan large batches of glass slides at high magnification and compress and store the large images on disc, which subsequently can be consulted through the Internet. The images are stored on an image server, which can give simple, easy to transfer pictures to the user specifying a certain magnification on any position in the scan. This offers new opportunities in histology review, overcoming the necessity of the dynamic telepathology systems to have compatible software systems and microscopes and in addition, an adequate connection of sufficient bandwidth. Consulting the images now only requires an Internet connection and a computer with a high quality monitor. A system of complete pathology review supporting bio-repositories is described, based on the implementation of this technique in the European Human Frozen Tumor Tissue Bank (TuBaFrost).

TuBaFrost 1: Uniting local frozen tumour banks into a European network: an overview.

TuBaFrost is the consortium responsible for the creation of a virtual European human frozen tumour tissue bank: a collection of high quality frozen residual, accurately classified tumour tissue samples, which are stored in European cancer centres and universities. This virtual tissue bank, searchable on the internet, has rules for access and use, and a code of conduct to comply with the various legal and ethical regulations in European countries. The easy accessibility and the European scale of the bank will result in the availability of a large number of samples even of rarer tumour types. Standardisation of collection, storage and quality control throughout the network is achieved minimising inter-institutional variability. A website providing access to upload, search and request samples is a key tool of the tissue bank. The search engine makes use of virtual microscopy. An overview of the development of the European virtual frozen tissue bank infrastructure is described in this paper. The various key aspects are described in more detail in a series of articles to appear in this Journal.

TuBaFrost 2: Standardising tissue collection and quality control procedures for a European virtual frozen tissue bank network.

Tumour Bank Networking presents a great challenge for oncological research as in order to carry out large-scale, multi-centre studies with minimal intrinsic bias, each tumour bank in the network must have some fundamental similarities and be using the same standardised and validated procedures. The European Human Frozen Tumour Tissue Bank (TuBaFrost) has responded to this need by the promotion of an integrated platform of tumour banks in Europe. The operational framework for TuBaFrost has drawn upon the best practice of standard workflows and operating procedures employed by members of the TuBaFrost project and key initiatives worldwide.

TuBaFrost 4: access rules and incentives for a European tumour bank.

When designing infrastructure for a networked virtual tumour bank (samples remain at the collector institutes and sample data are collected in a searchable central database), it is apparent that this can only function properly after developing an adequate set of rules for use and access. These rules must include sufficient incentives for the tissue sample collectors to remain active within the network and maintain sufficient sample levels in the local bank. These requirements resulted in a key TuBaFrost rule, stating that the custodianship of the samples remains under the authority of the local collector. As a consequence, the samples and the decision to issue the samples to a requestor are not transferred to a large organisation but instead remain with the collector, thus allowing autonomous negotiation between collector and requestor, potential co-authorship in publications or compensation for collection and processing costs. Furthermore, it realises a streamlined cost effective network, ensuring tissue visibility and accessibility thereby improving the availability of large amounts of samples of highly specific or rare tumour types as well as providing contact opportunities for collaboration between scientists with cutting edge technology and tissue collectors. With this general purpose in mind, the rules and responsibilities for collectors, requestors and central office were generated.

TuBaFrost 3: regulatory and ethical issues on the exchange of residual tissue for research across Europe.

The regulatory regimes for research with residual tissue and accompanying data differ widely between countries in the European Union (EU): from specific consent to opt-out or even no consent at all. This could greatly hamper research where the exchange of tissue and accompanying data has become the gold standard, like in TubaFrost. Instead of adhering to international guidelines, which have a democratic deficit, or an attempt for a new set of possible harmonising rules, TubaFrost chose to create a coordinating rule: if tissue may legitimately be used for a certain kind of research in the country where it was taken and under whose jurisdiction the patient falls, it may also be used for such research in the country where it is sent to in the context of a scientific program even if in that other country other regulations would apply for research with residual tissue taken from patients under their jurisdiction. This coordinating rule has a sound basis in EU law in general and will solve the problems related to diverging national regulatory regimes in the case of cross national research with residual tissue.

TuBaFrost 5: multifunctional central database application for a European tumor bank.

Developing a tissue bank database has become more than just logically arranging data in tables combined with a search engine. Current demand for high quality samples and data, and the ever-changing legal and ethical regulations mean that the application must reflect TuBaFrost rules and protocols for the collection, exchange and use of tissue. To ensure continuation and extension of the TuBaFrost European tissue bank, the custodianship of the samples, and hence the decision over whether to issue samples to requestors, remains with the local collecting centre. The database application described in this article has been developed to facilitate this open structure virtual tissue bank model serving a large group. It encompasses many key tasks, without the requirement for personnel, hence minimising operational costs. The Internet-accessible database application enables search, selection and request submission for requestors, whereas collectors can upload and edit their collection. Communication between requestor and involved collectors is started with automatically generated e-mails.

BMI-1: a protein expressed in stem cells, specialized cells and tumors of the gastrointestinal tract.

Recently, BMI-1 was identified as a protein downregulating p16ink4a and mandatory for the continued existence of several stem cell compartments like hematopoietic and neural stem cells. In this study we investigated BMI-1 expression as a potential stem cell marker of the gastrointestinal tract. We found weak expression in the isthmus region of the stomach, and moderate expression in crypts of the intestines, whereas intestinal surface epithelial cells were weakly positive or negative for BMI-1. In addition, a variety of highly differentiated cells such as parietal cells, neuroendocrine cells of the pylorus, Paneth cells and a subset of goblet cells were moderately to strongly positive for BMI-1. Furthermore, we detected strong expression in gastrointestinal neoplasias. This expression pattern indicates a correlation of BMI-1 expression with gastrointestinal stem cells as well as numerous specialized cell types and points to a role of this protein in cellular differentiation in addition to that of stem cell maintenance. Besides, our results imply a role for BMI-1 in the tumorigenesis of gastrointestinal cancer.

Inhibitors of differentiation/DNA binding proteins Id1 and Id3 are regulated by statins in endothelial cells.

Id proteins (inhibitors of differentiation), which are involved in the control of cell cycle progression, can delay cellular differentiation and senescence and have been implicated in angiogenesis. The regulation of Id proteins in endothelial cells (ECs) by proangiogenic statins has not been investigated yet and remains unresolved. In this study, human dermal microvascular ECs (HDMECs) were stimulated with fluvastatin, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and serum in vitro. The regulation of Id1, Id3, p21, p27, and p53 and the phosphorylation of AKT was investigated by Western blotting. Id1 was up-regulated by fluvastatin and serum, but not by VEGF and HGF. Fluvastatin did not regulate p21 and p27, but down-regulated Id3 and p53 slightly. In contrast to VEGF and HGF, fluvastatin did not result in AKT phosphorylation, indicating that this pathway is not involved in the control of endothelial Id1 expression. These experiments demonstrate for the first time that Id1 can be up-regulated and p53 down-regulated by a statin in HDMECs. Regulation of these proteins in ECs may account for the proangiogenic effect of statins.

In situ detection of the mast cell proteases chymase and tryptase in human lung tissue using light and electron microscopy.

Scroll-rich, "mucosal" mast cells are the predominant human lung mast cell type. It has been proposed that these mast cells store tryptase but are mostly chymase deficient. We present a detailed immunolocalisation study of chymase and tryptase in lung specimens of eight patients. Using monoclonal antibody B7 in a conventional tissue processing method for light microscopy, chymase-positive mast cells were much fewer than tryptase-positive ones. However, they approached the number of tryptase-positive cells when optimised processing was used. Two different monoclonal antibodies, B7 and CC1, were used to visualise chymase in purified lung mast cells of two patients using ultrastructural immunogold labelling. Immunoabsorption controls demonstrated a reactivity of B7 with both tryptase and chymase, but indicated specificity of CC1 for chymase. On the ultrastructural level, all of more than 1,400 lung mast cells evaluated labelled for chymase. Reactivity was seen in cytoplasmic granules, cytoplasm and vesicles, but not elsewhere. Tryptase labelling using monoclonal antibody G3 was also present in all mast cells detected, and was retained in altered granules (=activated mast cells), where B7 labelling was sparse. The average labelling density was approximately sixfold higher than for chymase. In summary, chymase may be more abundant in human lung mast cells than hitherto thought.

The pattern of prion-related protein expression in the gastrointestinal tract.

Prion diseases or transmissible spongiform encephalopathies have been shown to be communicated by oral ingestion of the infectious agent. However, the exact route of transmission is still unknown. In order to better understand the pathophysiology of these diseases, it is crucial to identify cell types of peripheral tissues in which the infectious agent may propagate. Since expression of cellular prion protein (PrPc) is a prerequisite for prion replication, we determined the expression of PrPc in the mucosa of the gastrointestinal tract using immunohistochemistry. Expression of PrPc was negative or weak in the neck region of the gastric mucosa and moderate to strong in crypts of both the small and the large bowel. PrPc was found to be upregulated in the mucosa of patients with Helicobacter pylori gastritis. In contrast, PrPc staining appeared to be downregulated in patients with inflammatory disorders of the large bowel and it remained moderate to strong in inflammatory disorders of the small bowel. Our results support the notion that epithelial cells of the gastrointestinal tract may represent a possible target for prion entry and replication.

Cellular prion protein expressed by bovine squamous epithelia of skin and upper gastrointestinal tract.

Routes of transmission of bovine spongiform encephalopathy have not yet been determined. We show that bovine squamous epithelia of the skin and upper gastrointestinal tract express Prp(c), suggesting that these epithelia may be a target for prion entry and replication.

The cell death regulatory protein bak is expressed in endothelial cells in inflamed tissues and Is induced by IFN-gamma in vitro.

In the present report, we examined the endothelial expression of the anti- and pro-apoptotic proteins Bcl-2 and Bak in situ and in vitro. Endothelial cells (EC) in regular tissue of the bowel and the skin were essentially negative for both Bcl-2 and Bak. In contrast, EC within the walls of fistulas and abscesses in these organs stained distinctly for Bak, but remained Bcl-2-negative. In tissue culture both unstimulated human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells (HUVEC) expressed Bcl-2 and Bak constitutively. Exposure of EC to 200-1000 IU IFN-gamma downregulated Bcl-2 but upregulated Bak. This opposing regulation of Bcl-2 and Bak in vitro and the expression of Bak in EC adjacent to necrotic tissue areas suggests that this pro-apoptotic protein may play a decisive role in regulation of EC survival in vivo.

Expression of vascular endothelial growth factor receptor-3 and podoplanin suggests a lymphatic endothelial cell origin of Kaposi's sarcoma tumor cells.

Despite intensive research over the past decade, the exact lineage relationship of Kaposi's sarcoma (KS) tumor cells has not yet been settled. In the present study, we investigated the expression of two markers for lymphatic endothelial cells (EC), ie, vascular endothelial growth factor receptor-3 (VEGFR-3) and podoplanin, in AIDS and classic KS. Both markers were strongly expressed by cells lining irregular vascular spaces in early KS lesions and by tumor cells in advanced KS. Double-staining experiments by confocal laser microscopy established that VEGFR-3-positive and podoplanin-positive cell populations were identical and uniformly expressed CD31. By contrast, these cells were negative for CD45, CD68, and PAL-E, excluding their hemopoietic and blood vessel endothelial cell nature. Podoplanin expression in primary KS tumor lysates was confirmed by Western blot analysis. Both splice variants of VEGFR-3 were found in KS-tumor-derived RNA by RT-PCR. In contrast to KS tumor cells in situ, no expression of VEGFR-3 and podoplanin was detected in any of four KS-derived spindle cell cultures and in one KS-derived autonomously growing cell line (KS Y-1). Our findings that KS tumor cells express two lymphatic EC markers in situ strongly suggest that they are related to or even derived from the lymphatic EC lineage. Lack of these antigens on cultured cells derived from KS lesions indicates that they might not represent tumor cells that grow in tissue culture, but rather other cell types present in KS lesions.