Absence of high-risk human papilloma virus in p16 positive inverted sinonasal papilloma.

OBJECTIVES: Sinonasal inverted papilloma (SIP) is a relatively rare disease, and its etiology is not understood. It is characterized by locally aggressive growth and a strong tendency to recur despite its benign histology. AIMS: The aim of this study was to identify the presence of human papilloma virus (HPV) and its surrogate marker p16 in SIP tissue samples from a regional cohort. MATERIAL AND METHODS: Subjects were identified from our regional center cohort of 88 SIP patients treated between 1984-2014. From these subjects, 54 were included in this study. Of these, 53 biopsies were analyzed with PCR, and 54 samples were immunohistochemically stained for p16. DNA was extracted from histopathologically verified SIP. Genotype screening for 13 high risk-, 5 oncogenic and 6 low risk HPV types was performed using the PapilloCheck® HPV-screening test. RESULTS: HPV analysis was successful for 38 of 53 samples. Of the 38 successfully analyzed samples, only 2 samples were positive for HPV 11. Notably, p16 was present in the epithelia in all samples, and in the papilloma lesions in 37 samples. CONCLUSION: Since only 2 out of 38 SIPs were positive for HPV (type 11), and at the same time p16 was positive in epithelia in all samples and in 37 of 38 papilloma lesions of the samples, it is concluded that p16 cannot be used as a surrogate marker for high-risk HPV-infection in SIP. We are currently planning a prospective, multicenter study in order to increase the study power and in order to be able to better evaluate the clinical implications of HPV-and p16 in SIP.

p53 binds the mdmx mRNA and controls its translation.

MDMX and MDM2 are two nonredundant essential regulators of p53 tumor suppressor activity. MDM2 controls p53 expression levels, whereas MDMX is predominantly a negative regulator of p53 trans-activity. The feedback loops between MDM2 and p53 are well studied and involve both negative and positive regulation on transcriptional, translational and post-translational levels but little is known on the regulatory pathways between p53 and MDMX. Here we show that overexpression of p53 suppresses mdmx mRNA translation in vitro and in cell-based assays. The core domain of p53 binds the 5' untranslated region (UTR) of the mdmx mRNA in a zinc-dependent manner that together with a trans-suppression domain located in p53 N-terminus controls MDMX synthesis. This interaction can be visualized in the nuclear and cytoplasmic compartment. Fusion of the mdmx 5'UTR to the ovalbumin open reading frame leads to suppression of ovalbumin synthesis. Interestingly, the transcription inactive p53 mutant R273H has a different RNA-binding profile compared with the wild-type p53 and differentiates the synthesis of MDMX isoforms. This study describes p53 as a trans-suppressor of the mdmx mRNA and adds a further level to the intricate feedback system that exist between p53 and its key regulatory factors and emphasizes the important role of mRNA translation control in regulating protein expression in the p53 pathway.

Expression of p16 in squamous cell carcinoma of the mobile tongue is independent of HPV infection despite presence of the HPV-receptor syndecan-1.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is increasing in incidence, especially among young patients and preferably females. Infection with human papilloma virus (HPV) has been suggested as a cause of SCC in the head and neck, and the proportion of oropharyngeal cancers caused by HPV has steadily increased. METHODS: Samples from 109 patients with primary TSCC were analysed for the presence of HPV16 by in situ hybridisation and for expression of its surrogate marker p16 and the HPV receptor syndecan-1 by immunhistochemistry. RESULTS: No evidence of HPV16 DNA was observed in the tumours, although one-third showed p16 staining. There was no difference in the expression of the primary HPV receptor, syndecan-1, between TSCC and a group of tonsil SCC. CONCLUSION: Whereas p16 is expressed in some TSCCs, HPV16 is undetectable, therefore, p16 cannot be used as a surrogate marker for high-risk HPV-infection in this tumour. Despite presence of the HPV-receptor syndecan-1 in TSCC, HPV prefers the tonsillar environment. Lack of p16 associates with worse prognosis primarily in patients aged ⩽40 years with tongue SCC. The improved prognosis seen in p16-positive TSCC can be due to induction of a senescent phenotype or an inherent radiosensitivity due to the ability of p16 to inhibit homologous recombination repair.

Downregulation of miRNA-424: a sign of field cancerisation in clinically normal tongue adjacent to squamous cell carcinoma.

BACKGROUND: The overall survival for patients with squamous cell carcinoma of the tongue is low and the search for early diagnostic and prognostic markers is thus essential. MicroRNAs have been suggested as potential prognostic and diagnostic candidates in squamous cell carcinoma of head and neck in general. METHODS: On the basis of the known differences between sub-sites within the oral cavity, we investigated the expression and role of microRNA-424 in squamous cell carcinoma arising in tongue. MicroRNA levels were measured by qRT-PCR in both tissue and plasma samples. RESULTS: Levels of microRNA-424 were upregulated in tongue squamous cell carcinoma, but not in tumours originating from gingiva or floor of the mouth. Interestingly, microRNA-424 was downregulated in clinically normal tongue tissue next to tumour compared with completely healthy tongue, indicating that microRNA-424 could be a marker of field cancerisation in this tumour type. However, expression of microRNA-424 in a tongue-derived epithelial cell line revealed no significant changes in the expression profile of proteins and genes. CONCLUSIONS: Our patient data show that microRNA-424 alterations are a marker of field cancerisation specific for tongue tumourigenesis, which also could have a role in development of tongue squamous cell carcinoma.

Autoantibodies and decreased expression of the transcription factor ELF-3 together with increased chemokine pathways support an autoimmune phenotype and altered differentiation in lichen planus located in oral mucosa.

BACKGROUND: The pathogenesis of oral lichen planus (OLP), a chronic inflammatory disease, is not fully understood. It is known that OLP has autoimmune features, and it is suggested to be an autoimmune disease. ELF-3 is involved in differentiation of keratinocytes and deregulated in different tumours and inflammatory diseases. CXCR-3 and its ligands CXCL-10 and CXCL-11 are increased in autoimmune diseases and linked to Th-1 immune response. OBJECTIVES: To analyse and compare expression of ELF-3, CXCR-3, CXCL-10 and CXCL-11 in OLP lesions and controls in whole and microdissected epithelium. METHODS: Tissue biopsies from 20 patients clinically and histologically diagnosed with OLP and 20 healthy controls were studied using whole tissues or microdissected epithelium. By the use of qRT-PCR, mRNA levels of ELF-3, CXCR-3, CXCL-10 and CXCL-11 were studied. Western blot was used for analysis of ELF-3 protein expression. Sera from 19 OLP patients and 20 controls were analysed with ELISA in search for autoantibodies. Results The upregulation of CXCR-3, CXCL-10 and CXCL-11 found in OLP is similar to previous findings showing an autoimmune phenotype in lichen planus (LP) and lichen sclerosus. Decreased expression of the differentiation-related transcription factor ELF-3 was also seen in OLP lesions, and we further demonstrate presence of circulating autoantibodies against the ELF-3 protein in sera from 3 of 19 (16%) LP patients tested. CONCLUSIONS: On the basis of these findings, we confirm that OLP shows features of an autoimmune disease and suggest deregulated differentiation of keratinocytes to be one of the causes of the disease phenotype.

Scandinavian Fellowship for Oral Pathology and Oral Medicine: guidelines for oral pathology and oral medicine in the dental curriculum.

In Scandinavia, as in many European countries, most patients consult their general dentist once a year or more. This gives the dentist a unique opportunity and an obligation to make an early diagnosis of oral diseases, which is beneficial for both the patient and the society. Thus, the dentist must have knowledge of clinical symptoms, local and systemic signs and clinical differential diagnoses to make an accurate diagnosis. The dentist must be competent in selecting appropriate diagnostic tests, for example, tissue biopsy and microbiological samples, and conducting them correctly, as well as in interpreting test results and taking appropriate action accordingly. Furthermore, the dentist must be aware of diseases demanding multidisciplinary cooperation and be able to recognise his/her professional limitation, and to refer to other specialists when required. The dental curriculum changes over time as new approaches, treatments and diagnostic possibilities develop. Likewise, the role of the dentist in the community changes and may vary in different countries. As members of the Scandinavian Fellowship for Oral Pathology and Oral Medicine and subject representatives of oral pathology and oral medicine, we feel obliged to contribute to the discussion of how the guidelines of the dental curriculum support the highest possible standards of dental education. This article is meant to delineate a reasonable standard of oral pathology and oral medicine in the European dental curriculum and to guide subject representatives in curriculum development and planning. We have created an advisory topic list in oral pathology and oral medicine.

Altered expression of miR-21, miR-125b, and miR-203 indicates a role for these microRNAs in oral lichen planus.

BACKGROUND: Oral lichen planus (OLP), which is a chronic inflammatory disease of the oral mucosa with unknown etiology, affects about 2% of the population. MicroRNAs are small non-coding RNAs involved in normal processes such as development and differentiation as well as progression of human diseases. The aim of this study was to investigate the expression of miR-21, miR-125b, and miR-203 and to compare RNA levels of their potential targets, the tumor suppressor p53 and its relative p63, both known to be deregulated in OLP. METHODS: In biopsies from 20 patients with OLP and 20 age- and sex-matched healthy controls, epithelium was laser dissected and analyzed for the expression of miR-21, miR-125b, miR-203, p53, and p63 using qRT/PCR. RESULTS: Increased expression of miR-21 and miR-203, decreased expression of miR-125, and down-regulation of p53 and ΔNp63 RNA were seen in OLP compared to normal oral mucosa. When comparing microRNA expression to levels of p53 and p63 RNA, a significant negative correlation was seen between ΔNp63 and miR-203 and between miR-21 and p53, respectively. CONCLUSION: Results indicate a role for the studied microRNAs in changes seen in OLP.

Increased levels of COX-2 in oral lichen planus supports an autoimmune cause of the disease.

BACKGROUND: Oral lichen planus (OLP) is a chronic inflammatory disease for which the pathogenesis is not fully understood. OLP has autoimmune features and auto immunity has been suggested as a potential cause, whereas WHO has classified OLP as a premalignant condition. Association between chronic inflammation and cancer is known and chronic inflammation is one of the characteristics of OLP. A protein connected to inflammation and suggested to be involved in cancer development is cyclooxygenase-2 (COX-2) which can be inhibited by microRNA-26b (miR-26b). OBJECTIVE: The aim was to map levels of COX-2 and miR-26b in OLP lesions to see if there was any correlation between expression of COX-2 and its regulator miR-26b in OLP. METHODS: In biopsies from 20 OLP patients and 20 age and gender-matched controls laser- micro dissection of epithelium was performed. Quantitative RT-PCR, immunohistochemistry and Western blot were used in the analysis. RESULTS: Levels of COX-2 mRNA were significantly higher while levels of miR-26b were significantly lower in OLP lesions compared to controls. Using immunohistochemistry normal oral mucosa samples did not show any expression of COX-2 while OLP samples expressed the protein. No COX-2 protein was detectable with Western blot. CONCLUSION: Increased expression of COX-2 and decreased expression of miR-26b in OLP suggests both to play a role in OLP. COX-2 has been connected to both malignant development and autoimmunity but as malignant development of OLP is quite rare we suggest that the increased levels of COX-2 seen here support an autoimmune cause of the disease.

miRNA analysis of formalin-fixed squamous cell carcinomas of the tongue is affected by age of the samples.

Global miRNA expression arrays were used for analysis of 836 miRNAs in formalin-fixed paraffin-embedded samples from 21 tongue cancer patients and 8 controls. Samples had been stored for one to eleven years. Results separated tumour samples from controls, however, the largest variation was correlated to sample storage time, detectable already after one year. With the use of a linear regression model we could adjust for the storage-dependent effect, leading to the identification of 54 differentially expressed miRNAs in tongue cancer, compared to 16 when using standard normalization, including up-regulation of a novel miRNA, miR-424.

Scandinavian Fellowship for Oral Pathology and Oral Medicine: statement on oral pathology and oral medicine in the European Dental Curriculum.

BACKGROUND: For many years, dentists have migrated between the Scandinavian countries without an intentionally harmonized dental education. The free movement of the workforce in the European Union has clarified that a certain degree of standardization or harmonization of the European higher education acts, including the dental education, is required. As a result of the Bologna process, the Association for Dental Education in Europe and the thematic network DentEd have generated guidelines in the document 'Profile and Competences for the European Dentist' (PCD). This document is meant to act as the leading source in revisions of dental curricula throughout Europe converging towards a European Dental Curriculum. In order to render the best conditions for future curriculum revisions providing the best quality dentist we feel obliged to analyse and comment the outlines of oral pathology and oral medicine in the PCD. METHODS: The representatives agreed upon definitions of oral pathology and oral medicine, and competences in oral pathology and oral medicine that a contemporary European dentist should master. The competences directly related to oral pathology and oral medicine were identified, within the PCD. RESULTS: The subject representatives suggested eighteen additions and two rewordings of the PCD, which all were substantiated by thorough argumentation. PERSPECTIVES: Hopefully, this contribution will find support in future revisions of the PCD in order to secure the best quality dental education.

Increased expression of Smad proteins, and in particular Smad3, in oral lichen planus compared to normal oral mucosa.

BACKGROUND: Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa which the World Health Organisation (WHO) considers a premalignant condition. One step in malignant development is so called epithelial mesenchymal transition (EMT), a process whereby epithelial cells acquire mesenchymal characteristics. EMT occurs during embryogenesis and wound healing but also in some human diseases such as cancer and fibrosis. A factor known to induce EMT is transforming growth factor-ß (TGF-ß), which uses the Smad proteins as mediators for its signalling. TGF-ß is also often over-expressed in squamous cell carcinoma of the head and neck (SCCHN). METHODS: In the present study we mapped expression of Smad proteins in OLP lesions by immunohistochemistry, and compared to expression in normal and sensitive oral mucosa. The latter group of patients had developed SCCHN after shorter or longer periods of diffuse oral symptoms. The aim was to see if there were any signs of EMT related changes in the OLP lesions, as judged by changes in the TGF-ß pathway. CONCLUSION: Changes in the TGF-ß pathway related to EMT are seen in the very earliest stages of oral malignancy and become more severe as lesions progress.

DeltaNp63 isoforms regulate CD44 and keratins 4, 6, 14 and 19 in squamous cell carcinoma of head and neck.

The human p63 gene codes for multiple protein isoforms and is commonly over-expressed in squamous cell carcinoma of head and neck (SCCHN). This expression is predominantly of the DeltaN- and beta-isoforms, the former lacking the p53-related transactivation domain. p63 can activate or repress transcription of p53 and p73 target genes, but also has unique transcriptional targets and, unlike other p53 family members, is required for normal development and differentiation of squamous epithelia. We have identified novel targets of p63, using microarray analysis of SCCHN cells that stably over-express individual DeltaNp63 isoforms. All three isoforms induced expression of the cancer stem cell marker, CD44, with the DeltaNp63beta isoform showing strongest induction. Using chromatin immunoprecipitation, we were unable to show direct binding of p63 to the CD44 promoter, but found that p63 specifically increased expression of CD44 lacking variant exon 2. Each of the DeltaNp63 isoforms up-regulated expression of keratins 6A and 14 and down-regulated expression of keratins 4 and 19, in keeping with their expression patterns in SCCHN. The data strengthen the idea that p63 has key roles in regulating normal and abnormal differentiation processes through both induction and repression of genes with opposite functions. The identification of up-regulation and differential splicing of CD44 following p63 over-expression indicates roles in the regulation of adhesion, metastasis and the cancer stem cell phenotype.

Exploiting oxidative stress and signaling in chemotherapy of resistant neoplasms.

Neural crest tumors of childhood are particularly resistant to apoptosis induction by chemotherapeutic agents. Mechanisms of resistance include altered glutathione handling that accompanies up-regulation of Bcl-2 and its relatives. We have designed and tested in preclinical model systems approaches to this problem. These approaches include adjunctive use of oxygen radical-generating neurotransmitter analogs taken up by these neural crest tumor cells with scavenging (i.e., "rescue") agents that are selective for normal neural crest and the use of reduction-dependent prodrugs of apoptosis-inducing agents. Promising prototypes for these conceptual approaches include, respectively, adjunctive use of the oxygen radical generator, 6-hydroxydopamine, with the normal cell-selective antioxidant, Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), and use of the reduction-dependent chemotherapeutic prodrug neocarzinostatin.

Differential expression of p73alpha in normal ectocervical epithelium, cervical intraepithelial neoplasia, and invasive squamous cell carcinoma.

The TP73 gene is a member of the p53 family and through differential promoter usage and alternative splicing can encode a number of different isoforms that have distinct properties. p73 proteins are widely expressed in neural, epithelial, and hemopoietic cells and are proposed to have roles in the development and differentiation of various cell types and in tumorigenesis. The authors have developed a novel monoclonal antibody that is specific for p73alpha to study the expression of this individual isoform in normal and neoplastic cervical epithelium. In normal epithelium, p73alpha is restricted to nonproliferating cells at the base of the epithelium, whereas other p73 isoforms are found in the proliferative zones higher up in the epithelium. In cervical cancers, p73alpha expression is commonly lost, although other p73 isoforms are present at high levels. In particular, the authors found that invasive islands lose p73alpha expression when compared with the overlying intraepithelial lesion. These results show a tight regulation of p73 isoform expression in cervical epithelium and imply that different isoforms of p73 enhance or suppress neoplastic cell growth. These data raise the possibility that reactivation of p73alpha might be beneficial in cervical carcinoma. In addition, the absence of p73alpha in cervical cancer represents a potentially useful tool for the diagnosis of this disease.

Expression of p53, PCNA, Ki-67 and bcl-2 in relation to risk factors in oral cancer - a molecular epidemiological study.

A group of 133 primary oral squamous cell carcinomas were studied concerning a relationship between exposure factors and tumour biological parameters with a focus on the TP53 gene and p53 protein status. Tumours were evaluated using immunohistochemistry (IHC) for expression of p53, PCNA, Ki-67 and bcl-2 proteins. The TP53 gene was studied for mutations using PCR amplification of exons 5-9 and single strand conformation polymorphism (SSCP) analysis. The collected data were correlated to the exposure factors smoking, oral snuff, liquor, oral infections, dental factors, dental X-ray and iron deficiency. When compared with matched controls only oral infections, and reported HSV-infections in particular, gave statistically significant ORs (odds ratio) for all tumours (OR 8.0) as well as for the group of IHC p53 positive tumours (OR 12). No association between smoking and p53 positivity was found (OR 1.0).

Early events in Bcl-2-enhanced apoptosis.

Transfection of PC12 pheochromocytoma cells with bcl-2 potentiates apoptosis induced by the antimitotic agent, neocarzinostatin (NCS). The mechanism of potentiation involves caspase 3-dependent cleavage of Bcl-2 to its pro-apoptotic counterpart, but the cellular events proximal to caspase 3 activation in this system are not known. Two min after initiation of NCS treatment, Bax begins to translocate from cytosol to the mitochondria; the mitochondrial localization of Bax persists for 30 min after NCS treatment. At the same time, cytochrome C is released from the mitochondria to cytosol. The mitochondrial membrane potential exhibits differential change in mock- and bcl-2 -transfected PC12 cells. In mock-transfected PC12 cells, the mitochondrial membrane potential increases immediately, peaks at 15 min following initiation of NCS treatment, and drops thereafter. In contrast, in bcl-2 -transfected PC12 cells, the membrane potential drops immediately following NCS treatment. Caspase 9 is activated and peaks at 10 min in both mock- and bcl-2 transfected PC12 cells, however, the peak activity of caspase 9 is higher and caspase 9 activation lasts longer (30 min) after the treatment in bcl-2 transfectants. Not until 30 min after initiation of a 1 h treatment with NCS is Bcl-2 protein cleaved in bcl-2 -transfected cells. Thus, in bcl-2 -transfected cells, the mitochondrial membrane potential drops and cytochrome C is released from the mitochondria despite the presence of large amounts of intact mitochondrial Bcl-2. This makes it unlikely that cleavage of Bcl-2 is the only factor involved in potentiation of NCS-induced apoptosis by Bcl-2.

p53 expression in correlation to clinical outcome in patients with renal cell carcinoma.

OBJECTIVE: The aim of this study was to evaluate the role of p53 as prognostic factor in renal cell carcinoma (RCC) and its relation to clinicopathological factors. MATERIAL AND METHODS: The nuclear accumulation of p53 protein was determined by immunohistochemical analysis in RCC specimens from 90 patients and was correlated with clinical stage, grade, DNA ploidy, S-phase fraction and cancer-specific survival. RESULTS: p53 overexpression was observed in 17 of 90 (19%) tumours. There was a significant correlation to stage (p = 0.016) and grade (p = 0.020) but not to DNA ploidy or S-phase. Patients with high p53 immunoreactivity had shorter cancer-specific survival (p = 0.003) than those with normal p53 protein expression. This difference was found in papillary and chromophobe tumour types (p < 0.0001) but not in conventional RCC. CONCLUSIONS: In patients with RCC, significant correlations between p53 protein expression and tumour stage, grade and survival time were observed. For patients with chromophobe and papillary tumour types, but not in conventional RCC, p53 immunoreactivity gave prognostic information, suggesting that the prognostic differences in p53 immunoreactivity might be due to disparate genetic abnormalities in the different RCC types.

The p53 molecule and its prognostic role in squamous cell carcinomas of the head and neck.

Despite intense research, the 5-year survival rate for patients with squamous cell carcinoma of the head and neck (SCCHN) is still low. Several different factors have been studied in the search for one or more factors that give important prognostic information at the time of diagnosis. Many recent studies have focused on the TP53 tumour suppressor gene, analysing its gene status and protein status. When looking at p53 protein expression, using immunohistochemistry, no correlation to patient outcome has been seen for the whole group of SCCHN. However, a significant association between p53 expression and poor patient outcome was found when looking only at patients with laryngeal squamous cell carcinomas. Also, in oral premalignant lesions, expression of p53-positive cells in the suprabasal layers of the epithelium has been seen as an indication of impending malignant development. Concerning the prognostic significance of mutations in the TP53 gene, results differ. But when restricting analysis to tumours with mutations causing an obvious change in protein, TP53 mutation was found to be a strong and independent variable for prognosticating survival. This review article gives an up-to-date overview of the p53 molecule and evaluates its possible prognostic role in SCCHN. Today it is clear that the p53 pathway is very important in SCCHN biology and potentially in its treatment. The function and importance of a few other cell cycle proteins connected to p53 are also discussed.

Characterization of the expression pattern of p63 alpha and delta Np63 alpha in benign and malignant oral epithelial lesions.

The p53 homologue p63 is essential for ectodermal differentiation, such that p63-/- mice lack all squamous epithelia and teeth. The p63 gene expresses at least 6 different transcripts, but information regarding the expression, regulation and function of the different isoforms has remained sparse, due to the lack of adequate reagents directed specifically against the individual proteins. Here we characterize the expression of p63 alpha/delta Np63 alpha in benign and malignant lesions of the oral epithelium, using a specific antibody raised against a peptide derived from the C-terminus of p63 alpha, which does not cross-react with p53 or the other p53 homologue, p73. By immunohistochemical analysis, we show that these p63 isoforms are expressed in the nucleus of many cells. In normal and benign lesions, p63 alpha/delta Np63 alpha-expressing cells are mainly found suprabasally, whereas p53-expressing cells are restricted to the basal-cell layer. By RT-PCR, we show that delta Np63 alpha is the predominant isoform in cell lines from squamous-cell carcinomas of the head and neck, confirming our immunochemical observations. Our data are consistent with studies suggesting a role for p63 in the transit-amplifying population of epidermal cells. Over-expression of p63 alpha, and in particular the delta N form, was frequently seen in carcinomas. Taken together with previous analyses of p63 expression, our data suggest distinct roles for different p63 isoforms in the regulation of growth and/or differentiation of epithelial cells. Moreover, our data are compatible with the notion that p63 can act to promote neoplastic growth in the oral epithelium.