The role of miRNAs in human papilloma virus (HPV)-associated cancers: bridging between HPV-related head and neck cancer and cervical cancer.

This corrects the article DOI: 10.1038/bjc.2012.109.

Molecular profiling of tumour budding implicates TGFß-mediated epithelial-mesenchymal transition as a therapeutic target in oral squamous cell carcinoma.

Although tumour budding is an adverse prognostic factor for many cancer types, the molecular mechanisms governing this phenomenon are incompletely understood. Therefore, understanding the molecular basis of tumour budding may provide new therapeutic and diagnostic options. We employ digital image analysis to demonstrate that the number of tumour buds in cytokeratin-stained sections correlates with patients having lymph node metastases at diagnosis. The tumour bud count was also a predictor of overall survival, independent of TNM stage. Tumour buds and paired central tumour areas were subsequently collected from oral squamous cell carcinoma (OSCC) specimens, using laser capture microdissection, and examined with RNA sequencing and miRNA-qPCR arrays. Compared with cells from the central parts of the tumours, budding cells exhibited a particular gene expression signature, comprising factors involved in epithelial-mesenchymal transition (EMT) and activated TGFß signalling. Transcription factors ZEB1 and PRRX1 were up-regulated concomitantly with the decreased expression of mesenchymal-epithelial (MET) transcription factors (eg OVOL1) in addition to Krüppel-like factors and Grainyhead-like factors. Moreover, miR-200 family members were down-regulated in budding tumour cells. We used immunohistochemistry to validate five markers of the EMT/MET process in 199 OSCC tumours, as well as in situ hybridization in 20 OSCC samples. Given the strong relationship between tumour budding and the development of lymph node metastases and an adverse prognosis, therapeutics based on inhibiting the activation of TGFß signalling may prove useful in the treatment of OSCC.

Increasing incidence of base of tongue cancers from 2000 to 2010 due to HPV: the largest demographic study of 210 Danish patients.

BACKGROUND: We assessed the development in the number of new base of tongue squamous-cell carcinoma (BSCC) cases per year in eastern Denmark from 2000 to 2010 and whether HPV may explain any observable increased incidence. METHODS: We performed HPV DNA PCR and p16 immunohistochemistry analysis for all (n=210) BSCCs registered in the Danish Head and Neck Cancer Group (DAHANCA) and the Danish Pathology Data Bank, and genotyped all HPV-positive specimens with amplicon-based next-generation sequencing. RESULTS: The overall crude incidence of BSCCs increased significantly (5.4% per year) during the study period. This was explained by a significant increase in the number of HPV-positive BSCCs (8.1% per year), whereas the number of HPV-negative BSCCs did not increase significantly. The overall HPV prevalence was 51%, with HPV16 as the predominant HPV type. CONCLUSIONS: The increased number of HPV-positive BSCCs may explain the increasing incidence of BSCCs in eastern Denmark, 2000-2010.

Correlation between human papillomavirus and p16 overexpression in oropharyngeal tumours: a systematic review.

BACKGROUND: A significant proportion of squamous cell carcinomas of the oropharynx (OP-SCC) are related to human papillomavirus (HPV) infection and p16 overexpression. This subgroup proves better prognosis and survival but no evidence exists on the correlation between HPV and p16 overexpression based on diagnostic measures and definition of p16 overexpression. We evaluated means of p16 and HPV diagnostics, and quantified overexpression of p16 in HPV-positive and -negative OP-SCCs by mode of immunohistochemical staining of carcinoma cells. METHODS: PubMed, Embase, and the Cochrane Library were searched from 1980 until October 2012. We applied the following inclusion criteria: a minimum of 20 cases of site-specific OP-SCCs, and HPV and p16 results present. Studies were categorised into three groups based on their definition of p16 overexpression: verbal definition, nuclear and cytoplasmatic staining between 5 and 69%, and ≥70% staining. RESULTS: We identified 39 studies with available outcome data (n=3926): 22 studies (n=1980) used PCR, 6 studies (n=688) used ISH, and 11 studies (n=1258) used both PCR and ISH for HPV diagnostics. The methods showed similar HPV-positive results. Overall, 52.5% of the cases (n=2062) were HPV positive. As to p16 overexpression, 17 studies (n=1684) used a minimum of 5-69% staining, and 7 studies (n=764) used ≥70% staining. Fifteen studies (n=1478) referred to a verbal definition. Studies showed high heterogeneity in diagnostics of HPV and definition of p16. The correlation between HPV positivity and p16 overexpression proved best numerically in the group applying ≥70% staining for p16 overexpression. The group with verbal definitions had a significantly lower false-positive rate, but along with the group applying 5-69% staining showed a worse sensitivity compared with ≥70% staining. CONCLUSIONS: There are substantial differences in how studies diagnose HPV and define p16 overexpression. Numerically, p16 staining is better to predict the presence of HPV (i.e. larger sensitivity), when the cutoff is set at ≥70% of cytoplasmatic and nuclear staining.

The role of miRNAs in human papilloma virus (HPV)-associated cancers: bridging between HPV-related head and neck cancer and cervical cancer.

BACKGROUND: Although the role of human papilloma virus (HPV) in cervical squamous cell carcinoma (CSCC) is well established, the role in head and neck SCC (HNSCC) is less clear. MicroRNAs (miRNAs) have a role in the cancer development, and HPV status may affect the miRNA expression pattern in HNSCC. To explore the influence of HPV in HNSCC, we made a comparative miRNA profile of HPV-positive (HPV+) and HPV-negative (HPV-) HNSCC against CSCC. METHODS: Fresh frozen and laser microdissected-paraffin-embedded samples obtained from patients with HPV+/HPV- HNSCC, CSCC and controls were used for microarray analysis. Differentially expressed miRNAs in the HPV+ and HPV- HNSCC samples were compared with the differentially expressed miRNAs in the CSCC samples. RESULTS: Human papilloma virus positive (+) HNSCC had a distinct miRNA profile compared with HPV- HNSCC. Significantly more similarity was seen between HPV+ HNSCC and CSCC than HPV- and CSCC. A set of HPV core miRNAs were identified. Of these especially the miR-15a/miR-16/miR195/miR-497 family, miR-143/miR-145 and the miR-106-363 cluster appear to be important within the known HPV pathogenesis. CONCLUSION: This study adds new knowledge to the known pathogenic pathways of HPV and substantiates the oncogenic role of HPV in subsets of HNSCCs.

Different miRNA signatures of oral and pharyngeal squamous cell carcinomas: a prospective translational study.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs, which regulate mRNA translation/decay, and may serve as biomarkers. We characterised the expression of miRNAs in clinically sampled oral and pharyngeal squamous cell carcinoma (OSCC and PSCC) and described the influence of human papilloma virus (HPV). METHODS: Biopsies obtained from 51 patients with OSCC/PSCC and 40 control patients were used for microarray analysis. The results were correlated to clinical data and HPV status. Supervised learning by support vector machines was employed to generate a diagnostic miRNA signature. RESULTS: One hundred and fourteen miRNAs were differentially expressed between OSCC and normal oral epithelium, with the downregulation of miR-375 and upregulation of miR-31 as the most significant aberrations. Pharyngeal squamous cell carcinoma exhibited 38 differentially expressed miRNAs compared with normal pharyngeal epithelium. Differences in the miRNA expression pattern of both normal epithelium and SCC were observed between the oral cavity compared with the pharynx. Human papilloma virus infection revealed perturbations of 21 miRNAs, most significantly in miR-127-3p and miR363. A molecular classifier including 61 miRNAs was generated for OSCC with an accuracy of 93%. CONCLUSION: MicroRNAs may serve as useful biomarkers in OSCC and PSCC. The influence of HPV on miRNA may provide a mechanism for the distinct clinical behaviour of HPV-infected tumours.

Expression of two drug-metabolizing cytochrome P450-enzymes in human salivary glands.

OBJECTIVE: The oral cavity is constantly lubricated by saliva and even small amounts of xenobiotics and / or their metabolites in the saliva may affect the oral mucosa. Our aim was therefore to clarify if xenobiotic metabolizing enzymes CYP1A2 and CYP3A4 are expressed in salivary glands. METHODS: Formalin-fixed paraffin-embedded specimens from parotid (10), submandibular (7) and labial (10) salivary glands were examined immunohistochemically and by in situ hybridization for expression of CYP1A2 and CYP3A4 protein and mRNA. RESULTS: CYP1A2 and CYP3A4 protein and mRNA were detected in ductal and seromucous / serous acinar cells in all gland types although to a varying degree and intensity. Mucous acinar cells were positive to a lesser extent. CONCLUSION: The results indicate a xenobiotic metabolizing capability of salivary glands. This may have implications for development of oral mucosal disease as a result of mucosal exposure to metabolites originating from internal sources (blood) as well as from saliva.

Loss of a novel mucin-like epithelial glycoprotein in oral and cervical squamous cell carcinomas.

Stratified squamous epithelia of oral and cervical mucosa express high levels of simple mucin-type O-linked carbohydrates, and these are known to undergo structural changes in relation to epithelial differentiation and neoplastic transformation. O-glycans in these epithelia are associated with the cell membrane, but the identity of the carrier molecule(s) remains largely unknown. We report here the identification of a membrane-bound M(r) 200,000-250,000 glycoprotein (gp230) that is expressed in stratified squamous epithelia of the oral cavity. Western blot analysis identified gp230 as a major carrier of simple-mucin type carbohydrate antigens in buccal nonkeratinized mucosal epithelium, suggesting that it may represent a mucin-like molecule. A monoclonal antibody PANH4 defining a protein epitope of gp230 was generated. The PANH4 epitope was localized by immunohistology to suprabasal cell layers of buccal epithelium and was also found in larynx, esophagus, vagina, and exocervix, but not in epidermis. Data showed that gp230 was distinct from MUC1 or CD44. It is interesting that in most cases gp230 was not expressed in squamous cell carcinomas of buccal and cervical mucosa. A few moderately differentiated carcinomas, mainly from cervix, expressed the gp230 epitope. The results suggest that a membrane-bound mucin-like molecule, gp230, is associated with the differentiated phenotype of normal mucosal stratified squamous epithelia and that expression of gp230 generally is lost in severe oral epithelial dysplasia and squamous cell carcinomas of oral and cervical mucosa.

Simple mucin-type carbohydrate antigens in major salivary glands.

Simple mucin-type carbohydrate antigens Tn, sialosyl-Tn and T are often markers of neoplastic transformation and have very limited expression in normal tissues. We performed an immunohistological study of simple mucin-type carbohydrate antigens, including H and A variants, with well-defined monoclonal antibodies (MAb) on frozen and paraffin-embedded normal salivary gland tissue from 22 parotid, 14 submandibular, six sublingual, and 13 labial glands to elucidate the simple mucin-type glycosylation pattern in relation to cyto- and histodifferentiation. The investigated carbohydrate structures were predominantly observed in the cell cytoplasm, most often in the supranuclear area, suggesting localization to the Golgi region, whereas ductal contents were unstained. Mucous acinar cells expressed Tn, sialosyl-Tn, and H and A antigens, regardless of glandular location. Serous acinar cells, on the other hand, expressed A, H, and inconstantly sialosyl-T, Tn, and sialosyl-Tn antigens in major salivary glands, whereas serous cells of minor (labial) salivary glands expressed H exclusively, Tn and sialosyl-T antigens inconstantly, but never sialosyl-Tn and A antigens. The difference may be related to a more simple cytodifferentiation of serous cells of minor (labial) salivary glands as compared with major salivary glands. Duct cells in major salivary glands expressed A, H, and inconstantly T, sialosyl-T, and Tn antigens, whereas minor (labial) salivary glands ducts exclusively expressed H, T and sialosyl-T antigens, differences that may be related to dissimilarities in the duct system. Myoepithelial cells and basal cells exclusively expressed T and sialosyl-T antigens, which may prove useful in studies of salivary gland tumors, since these cells are known to play a key role in the histological characteristics of some salivary gland tumors. The results indicate a similar glycosylation pattern in the different major salivary glands, whereas minor (labial) salivary gland differ slightly in serous and duct cells. The limited and exclusive intracellular expression of the immature Tn, sialosyl-Tn, and T antigens indicates that these structures may be of value as markers of salivary gland tumors.

Undifferentiated carcinoma of the salivary gland in Greenlandic Eskimos: demonstration of Epstein-Barr virus DNA by in situ nucleic acid hybridization.

Paraffin sections of 11 undifferentiated salivary gland carcinomas of lymphoepithelioma type (malignant lymphoepithelial lesion) arising in Greenlandic Eskimos (Inuit) were examined for the presence of Epstein-Barr virus (EBV) using in situ nucleic acid hybridization with a 35S-labeled EBV-specific probe. Epstein-Barr virus genomes were detected in each case in malignant epithelial cells, but were not found in lymphoid stroma or in residual benign salivary epithelium. Eight undifferentiated salivary gland carcinomas from non-Eskimo patients (including two with lymphoepithelioma-like features) were negative for EBV-DNA. Our results confirm the existence of a consistent and specific association between EBV and tumor cells of undifferentiated salivary gland carcinoma of lymphoepithelioma type arising in Greenlandic Eskimos.

Microwave-assisted frozen section diagnosis. A comparison between conventional cryostat technique and the combination of freezing and microwave-stimulated fixation.

The study evaluates the morphology of frozen tissue sections treated by microwave-stimulated fixation compared with unfixed cryostat sections. Furthermore, microwave fixation has been compared with fixation at room temperature. Improved preservation of nuclear structures (chromatin pattern and nucleoli) was found with the microwave combination, while other parameters were indistinguishable from the unfixed cryostat sections. Fixation at room temperature showed no difference compared with the unfixed cryostat sections. The combined method might be useful in rapid diagnosis of mesenchymal tumors, lymphoid proliferations and tumors of the CNS.

Observer variability in histological malignancy grading of adenoid cystic carcinomas.

The value of malignancy grading of adenoid cystic carcinomas (ACC) is controversial. Some studies have shown that tumours with a solid growth component have a rapid fatal course, compared to tumours without a solid growth component, in which recurrences develop even many years after initial treatment. Other studies have failed to correlate growth patterns with clinical course. No universally accepted grading system exists and no reproducibility studies of the existing grading systems have been performed. The aim of this study was to examine the reproducibility of grading based on semi-quantitative assessment of the solid growth pattern in ACC. Two different grading systems were assessed by 3 observers on a material of 59 ACC. Interobserver agreement was evaluated using the kappa statistic. The reproducibility of grading was poor, except for the category "solid component constituting 50% or more of the tumour" (kappa = 0.52). It is concluded that quantitative methods are necessary if grading is to be used in prognostic evaluation of ACC. The rarity of the tumours, however, combined with difficulties in diagnosis will impede such investigations unless multicentre studies are undertaken.

A case of granulomatous sialadenitis of the submandibular gland.

Granulomatous inflammation of the major salivary glands is very rare and may be due to obstruction. Little attention has been paid to this condition. The reaction is caused by extravasation of mucus, as seen in the common mucocele of the minor salivary glands. A case of granulomatous inflammation of the submandibular gland caused by obstruction is presented. The etiology of granulomatous sialadenitis is reviewed.

Simple mucin-type carbohydrate antigens in pleomorphic adenomas.

Simple mucin-type carbohydrate structures, T, Tn and sialosyl-Tn, are regarded as general markers of carcinomas in several epithelial tissues as a result of incomplete synthesis with precursor accumulation. The structures have a very limited distribution in normal tissues and secretions, including saliva and salivary glands. The expression of simple mucin-type carbohydrate structures and ABH(O) variants was studied in paraffin-embedded and frozen tissue sections from 37 pleomorphic adenomas with associated normal parotid tissue, using immunohistology and a panel of MAbs with well-defined specificity for T, Tn, sialosyl-Tn, and blood group H and A variants hereof. The immature Tn and sialosyl-Tn antigen structures were expressed in the epithelial ductular structures of the tumors, whereas they were almost absent from normal parotid tissue, indicating aberrant glycosylation with accumulation of precursor structures. Furthermore, the tumors showed loss of A antigen. The prognostic significance of these results is discussed. The modified myoepithelial cells in periductular and solid areas expressed T and sialosyl-T antigens, similar to normal myoepithelial cells and basal cells. Thus these modified MEC seem to have retained their normal simple mucin-type glycosylation pattern, suggesting that T antigen may be used as a marker of MEC in salivary gland tumors.

Thomsen-Friedenreich (T) antigen as marker of myoepithelial and basal cells in the parotid gland, pleomorphic adenomas and adenoid cystic carcinomas. An immunohistological comparison between T and sialosyl-T antigens, alpha-smooth muscle actin and cytokeratin 14.

Controversy centres on the role and identification of myoepithelial (MEC) and basal cells in salivary gland tumours, and recent studies suggest that both basal cells and myoepithelial cells participate in the formation of salivary gland tumours. We have correlated the expression of different well-known markers of normal MEC/basal cells (i.e. alpha-smooth muscle actin and cytokeratin 14) with T (Thomsen-Friedenreich) antigen and its sialylated derivative: sialosyl-T antigen,) in 17 normal parotid glands and in two tumour types with MEC participation (i.e pleomorphic adenomas (PA) and adenoid cystic carcinomas (ACC)) using immunohistology with well-defined monoclonal antibodies (MAbs). Paraffin-embedded/fresh frozen tissue sections were studied from 33/17 patients with PA and 15/7 patients with ACC. In normal parotid tissue coexpression of alpha-smooth muscle actin, cytokeratin 14, T and sialosyl-T antigens was found in all MEC and in some of the basal cells lining striated ducts. The remaining basal cells exclusively expressed cytokeratin 14, T and sialosyl-T antigens. In the tumours, cells believed to be modified myoepithelial cells showed two different staining patterns: 1) Coexpression of alpha-smooth muscle actin, cytokeratin 14, T and sialosyl-T antigens, and 2) Coexpression of cytokeratin 14, T and sialosyl-T antigens, but no alpha-smooth muscle actin. The epithelial ductular structures in the tumours showed aberrant expression of cytokeratin 14, T and sialosyl-T antigens, and cytokeratin 14 was the only marker of cells in solid undifferentiated areas of adenoid cystic carcinomas. Our study supports the view, that modified "myoepithelial" cells in the tumours consist of a mixture of basal cells and myoepithelial cells. None of the investigated structures was in itself an ideal marker in the identification of MEC/basal cells. The cells can be identified by a combination of markers (i.e. cytokeratin 14, alpha-smooth-muscle actin, T and sialosyl-T antigens).

Altered expression of ABO (H) carbohydrate antigens is seen in pleomorphic adenomas.

Cell surface carbohydrate antigens show changes in relation to differentiation, maturation and malignant transformation. The expression of type 2 chain ABH carbohydrate structures of the ABO histo-blood group system was investigated in 28 pleomorphic adenomas (PA) and normal parotid glands in order to study possible changes in the glycosylation pattern. The distribution of carbohydrate structures was investigated by immunohistological stainings of formalin-fixed paraffin-embedded material using monoclonal antibodies (MAbs) with well-defined specificity. A strong interindividual variation was found in the normal tissue as well as in the tumors. In normal tissue, acinus and duct cells all expressed elongated carbohydrate structures. The yoepithelial cells did not stain with any of the MAbs investigated. In the PAs, staining was seen in the ductular structures and myoepithelial cells. In contrast to normal tissue, the tumors expressed the short precursor molecule sialylated N-acetyllactosamine. Furthermore, the PAs showed loss of H and A antigens, and a reduced expression of Le(y) compared to normal tissue. The ductular structures as well as the modified myoepithelial cells expressed binary N-acetyllactosamine, which in the normal tissue could only be found in the striated and excretory ducts. Thus our study has shown that aberrant glycosylation is not only a feature of malignant neoplasms but also occurs in pleomorphic adenomas.

Oncofetal fibronectins in oral carcinomas: correlation of two different types.

Different isoforms of fibronectin are derived from a single gene by alternative processing of the primary RNA transcript or by posttranslational modifications. We have previously demonstrated that an oncofetal fibronectin (FN) isoform derived by O-glycosylation is highly associated with malignancy in breast and oral tumors. Another oncofetal FN isoform containing the ED-B sequence is derived by alternative splicing, and FN containing ED-B has been found to be a stromal marker of malignancies in various tissues. Here we report a comparative study by immunohistology of the distribution of the ED-B-containing isoform and the oncofetal FN isoform derived by O-glycosylation, in oral squamous cell carcinomas, premalignant lesions, and normal oral mucosa. A selective expression of the ED-B-containing isoform was demonstrated in close relation to the invading carcinoma (38/38), whereas there was virtually no staining in submucosa underlying premalignant lesions (1/11) and normal epithelium (0/5). The ED-B-containing FN showed close co-distribution and staining pattern with the oncofetal isoform derived by O-glycosylation. These results demonstrate that accumulation of FN adjacent to oral carcinomas includes both the ED-B-containing isoform and the isoform derived by O-glycosylation. Although both the change in primary structure and glycosylation of FN create conformational and immunologically detectable changes, the functional consequences in association with invasive carcinoma are poorly understood at present. Diagnostic implications especially of borderline lesions as well as evaluation of tumor aggressiveness may, however, be important.

Can alterations in integrin and laminin-5 expression be used as markers of malignancy?

Development of squamous cell carcinomas (SCCs) involves alterations in the adhesive interactions in the epithelium and invasion through the basement membrane. Therefore, changes in the expression of receptors and ligands involved in cell-cell and cell-matrix adhesion may be essential for the transformation of a premalignant into a malignant lesion. The aim of this study was to examine if expression of specific cell adhesion molecules can be used as markers of malignant development. By immunohistochemistry, we examined the expression pattern of integrins alpha2beta1, alpha3beta1, alpha6beta4 and laminin-5 in biopsies from SCCs (n=18), premalignant lesions (leukoplakias, n=21) and non-premalignant tissue with chronic inflammation (n=11). In poorly differentiated SCCs, patchy loss of alpha3beta1, alpha6beta4 and laminin-5 expression was pronounced at the invasion front, whereas there was a tendency to increased expression of alpha2beta1. Analogous to the SCCs, biopsies from the leukoplakias and the non-premalignant inflammatory tissue showed alterations of the expression of alpha3beta1 and alpha6beta4 in the basal cell layers and of laminin-5. However, a characteristic finding in biopsies from leukoplakias was loss of alpha2beta1 and alpha3beta1 in the suprabasal cells. There was no unequivocal expression of the adhesion molecules distinguishing between inflammatory tissue, premalignant, and malignant lesions.

Differential expression of human high-molecular-weight salivary mucin (MG1) and low-molecular-weight salivary mucin (MG2).

Two distinct mucin components of saliva, MG1 and MG2, have been identified based on chemical composition and molecular weights (high and low, respectively) in saliva. With the aim of characterizing the expression pattern of salivary mucins, we have prepared monoclonal antibodies (MAbs) directed against the peptide core of MG1 and against a synthetic peptide derived from the MG2 (MUC7) sequence. MAb PANH2 raised against partially deglycosylated MG1 stained a high-molecular-weight smear in Western blots of partially purified MG1. PANH2 binding was increased by deglycosylation with trifluoromethanesulfonic acid as well as with subsequent periodate treatment, and was eliminated by pronase treatment, strongly suggesting that MAb PANH2 was directed to a peptide epitope of MG1. MAb PANH3 raised against a synthetic peptide derived from the MG2 (MUC7) sequence reacted with the native molecule and stained a narrow smear of ca. 200,000 to 210,000 in Western blots of concentrated saliva and a lower-molecular-weight smear of trifluoromethanesulfonic-acid-treated MG2. Immunohistology on frozen sections of human salivary glands showed that MAb PANH2 selectively labeled mucous cells, whereas MAb PANH3 labeled subpopulations of serous cells. Double-direct immunofluorescence staining with PANH2 and PANH3 demonstrated that the staining patterns were non-overlapping. The development of these antibody probes will facilitate studies of mucin expression in diseases of salivary glands.

Salivary gland carcinomas: prognostic significance of simple mucin-type carbohydrate antigens.

The prognosis of salivary gland carcinomas is difficult to assess. Simple mucin-type carbohydrates (T and sialosyl-T antigens, Tn and sialosyl-Tn antigens) have been shown to be of value in predicting prognosis for carcinomas in other locations. We studied the prognostic significance of the expression of these structures in a retrospective study of 133 patients with salivary gland carcinomas, using immunohistochemistry and a panel of well-defined monoclonal antibodies (MAbs) on formalin-fixed paraffin-embedded tissues. Sialosyl-Tn, T and sialosyl-T antigens were not correlated with prognosis. Univariate analyses showed no overall difference in survival or locoregional control between patients with Tn-positive and patients with Tn-negative tumours, but indicated that expression of the Tn antigen was associated with early locoregional recurrences and deaths. Tn was, however, not an independent prognostic factor by multivariate regression analysis.