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.

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 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 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 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.

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).

Early onset of pulmonary mucormycosis with pulmonary vein thrombosis in a heart transplant recipient.

Pulmonary infections are major complications in heart transplantation. Progress in antimicrobial chemotherapy has switched the clinical spectrum to an increased incidence of fungal pathogens, such as Candida and Aspergillus species. Mucormycosis is a rare opportunistic infection with high mortality in solid-organ transplant recipients usually ensuing several months after transplantation. We describe a 45-year-old patient with pulmonary mucormycosis manifestion 5 days after heart transplantation. The infection resulted in pulmonary vein thrombosis followed by hemorrhagic infarction. Despite antifungal treatment and surgical resection, the patient died on day 14 after transplantation. Antemortem diagnostic procedures were negative; autopsy confirmed the presence of Rhizopus oryzae invading blood vessels. We conclude that physicians must be aware of mucormycosis even within one week after heart transplantation--which has not been described so far. Invasive diagnostic workup is mandatory in case of suspicion; amphotericin B and, in selected cases, surgical resection are the mainstays of therapy.

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.

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.

Expression of bcl-2 in salivary glands and salivary gland adenomas. A contribution to the reserve cell theory.

The bcl-2 proto-oncogene product inhibits apoptosis. It has been suggested that bcl-2 assists the survival of stem cells. Bcl-2 also plays a role in the development of adenomas. In salivary glands it is expressed in basal cells of striated and excretory ducts which may indicate that these cells are reserve cells. Acinar cells, myoepithelial cells and most luminal cells are negative for bcl-2. In basal cell adenomas and Warthin's tumors it is found predominantly in cells with basal cell differentiation. In pleomorphic adenomas bcl-2 is expressed mainly in basal cells of tubulo-ductal structures, at various degrees and patterns in solid and trabecular areas and at low degree in myxoid areas. In chondroid areas of pleomorphic adenomas, in myoepitheliomas and oncocytomas it is only focally expressed or missing. If the inhibition of apoptosis plays a significant role in the genesis of these neoplasms, then factors other than bcl-2 must be effective.

Expression of bcl-2, bcl-x, bax and bak in renal parenchyma, oncocytomas and renal cell carcinomas.

Proteins of the bcl-2 family are important regulators of programmed cell death. Alterations in the expression of these proteins may contribute to the progression of cancer. Expression of bcl-2, bcl-x, bax and bak was investigated by immunohistochemistry and Western-blotting of regular and alterated renal parenchyma as well as in 57 renal cell carcinomas. Bcl-2, bcl-x and in part bax were found to be overexpressed in inflammed renal parenchyma, whereas atrophic tubuli predominantly stained for bcl-2 and to a lesser degree for bcl-x and bax. Only little bak expression was detected in alterated tubuli. Moderate to strong expression for bcl-2, bcl-x, bax and bak was found in 24, 38, 2 and 13 of 57 carcinomas, respectively. Bcl-2, bcl-x, bax and bak expression were correlated to tumor type. Chromophilic carcinomas stained stronger for bcl-2, bcl-x and bax, whereas chromophobic carcinomas stained stronger for bcl-x, bax and bak compared to clear cell carcinomas. Expression of bak correlated with that of bcl-x and with an unfavorable histology as indicated by nuclear grading in these tumors. Our findings suggest that expression of bcl-2 and bcl-x may be important for cell survival only in a subset of renal cell carcinomas, and that the anti-apoptotic effect of these proteins appears to be frequently bypassed possibly by other factors impeding programmed cell death.

[Mucormycosis--a rare complication in patients with diabetes mellitus]. / Mucormykose--Eine seltene Komplikation bei Patienten mit Diabetes mellitus.

Mucormycosis usually occurs in immunocompromised patients or in patients with diabetes mellitus. Pathogens are moulds of the mucorales species. The diagnosis is made by histological examination of biopsies. A 39 year-old patient with insulin-dependent diabetes mellitus was admitted with a tentative diagnosis of a tumour of the maxilla. After diagnosis of hyphae of the mucorales species, the patient's diabetes was stabilised and he was treated over 17 weeks with amphotericin B (40 mg per day) and made a good recovery. A 58 year-old insulin-dependent patient with ethmoidali and sphenoidali sinusitis did not respond to antibiotic therapy. Mucormycosis was diagnosed by means of biopsy. Although treatment with amphotericin B was started, the patient died after 3 weeks due to multiple organ failure.

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.

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.