Penile Cancers Attributed to Human Papillomavirus Are Associated with Improved Survival for Node-positive Patients. Findings from a Norwegian Cohort Study Spanning 50 Years.

BACKGROUND: Human papillomavirus (HPV) infection is a risk factor for the development of penile squamous cell carcinoma (PSCC). It remains inconclusive whether HPV-related PSCC has a different prognosis from non-HPV-related PSCC. OBJECTIVE: To investigate the relationship between HPV status and survival as well as temporal changes in the proportion of HPV-related PSCC. DESIGN, SETTING, AND PARTICIPANTS: A retrospective cohort of 277 patients treated in Norway between 1973 and 2022 was investigated for HPV DNA in tumor tissue. Clinicopathological variables and disease course were registered. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Kaplan-Meier curves and Cox regression were used to investigate the determinants of cancer-specific survival (CSS). The chi-square test for trend in proportions enabled investigation of temporal changes in the HPV-related proportion of PSCC patients treated in Western Norway (n = 211). RESULTS AND LIMITATIONS: HPV DNA was detected in tumor tissue from 131 (47%) patients. Stratified by HPV status, 5-yr CSS did not differ between groups (p = 0.37). When investigating only node-positive patients, however, presence of HPV DNA was an independent predictor of better survival in multivariable Cox regression after adjustment for age, nodal stage, and adjuvant therapy (hazard ratio 0.54, 95% confidence interval: [0.30-0.99], p = 0.04). In cases from Western Norway, an increasing proportion of HPV-related cases over time was found (p = 0.01). The main limitation is the retrospective study design. CONCLUSIONS: HPV DNA in tumor tissue was associated with significantly better CSS for node-positive patients. The proportion of HPV DNA-positive PSCC has increased significantly in Western Norway over the past 50 yr. PATIENT SUMMARY: We investigated the impact of human papillomavirus (HPV) on the survival of penile cancer patients treated over a 50-yr period in Norway. We found that for patients with lymph node metastasis, survival was better for HPV-related cases. We also found that the proportion of cases due to HPV has increased in Western Norway.

Loss of S100A14 expression at the tumor-invading front correlates with poor differentiation and worse prognosis in oral squamous cell carcinoma.

BACKGROUND: We previously showed a tumor-suppressive function of S100A14 in oral squamous cell carcinoma (OSCC). This study aimed to examine the prognostic significance and differentiation-related function of S100A14 in OSCC. METHODS: S100A14 expression was examined in 170 OSCCs from Norwegian and Nepalese populations using immunohistochemistry. Pro-differentiation function was investigated by overexpressing and silencing S100A14 expression in OSCC-derived cells. External transcriptomic datasets were used to validate association between S100A14 and differentiation markers in OSCC. RESULT: Loss of S100A14 expression at the invading tumor fronts significantly correlated with poor differentiation and reduced 10-years survival of OSCC-patients. Multivariate Cox analysis identified S100A14 to be an independent prognostic factor. Modulation of S100A14 expression in OSCC-derived cells positively correlated with the expression of differentiation markers. Analysis of external datasets supported the pro-differentiation function of S100A14. CONCLUSION: These results indicate that S100A14 is a pro-differentiation protein and its expression might be useful as a prognostic marker in OSCC.

Impact of humanised isolation and culture conditions on stemness and osteogenic potential of bone marrow derived mesenchymal stromal cells.

Therapeutic potential of human bone marrow stromal/stem cells (hBMSC) must be developed using well defined xenogenic-free conditions. hBMSC were isolated from healthy donors (n = 3) using different isolation and expansion methods. Donor I was isolated and expanded by either bone marrow directly seeded and cells expanded in 10% AB human serum (AB) +5 ng/ml fibroblast growth factor-2 (FGF2) [Direct(AB + FGFlow)] or Ammonium-Chloride-Potassium Lysing Buffer was used before the cells were expanded in 10% AB +5 ng/ml FGF-2 [ACK(AB + FGFlow)] or Lymphoprep density gradient medium was used before the cells were expanded in 10% AB +5 ng/ml FGF2 [Lympho(AB + FGFlow)] or bone marrow directly seeded and cells expanded in 10% pooled platelet lysate plasma (PL) + heparin (2 I/U/mL) [Direct(PL)]. Groups for donors II and III were: Direct(AB + FGFlow) or 10% AB +10 ng/ml FGF2 [Direct(AB + FGFhigh)] or Direct(PL). HBMSCs were assessed for viability, multi-potency, osteogenic, inflammatory response and replicative senescence in vitro after 1 and 3 weeks. Pre-selected culture conditions, Direct(AB + FGFhigh) or Direct(PL), were seeded on biphasic calcium phosphate granules and subcutaneously implanted in NOD/SCID mice. After 1 and 11 weeks, explants were analysed for inflammatory and osteogenic response at gene level and histologically. To identify implanted human cells, in situ hybridisation was performed. hBMSC from all conditions showed in vitro multi-lineage potency. hBMSCs expanded in PL expressed stemness markers in vitro at significantly higher levels. Generally, cells expanded in AB + FGF2 conditions expressed higher osteogenic markers after 1 week both in vitro and in vivo. After 11 weeks in vivo, Direct(AB + FGFhigh) formed mature ectopic bone, compared to immature mineralised tissues formed by Direct(PL) implants. Mouse responses showed a significant upregulation of IL-1α and IL-1ß expression in Direct(PL). After 1 week, human cells were observed in both groups and after 11 weeks in Direct(AB + FGFhigh) only. To conclude, results showed a significant effect of the isolation methods and demonstrated a relatively consistent pattern of efficacy from all donors. A tendency of hBMSC expanded in PL to retain a more stem-like phenotype elucidates their delayed differentiation and different inflammatory expressions.

Effect of glycerol on reconstructed human oral mucosa.

The majority of severely ill patients experience dry mouth. For institutionalized patients, this condition is commonly treated using glycerol as a lubricant. However, because of its possibly desiccating effect, some countries do not advocate the use of glycerol. This study aimed to investigate dose-dependent effects of glycerol on homeostasis and tissue integrity of in vitro-reconstructed normal human buccal mucosa (RNHBM). Primary keratinocytes and fibroblasts were isolated and expanded from biopsies of mucosa from eight healthy volunteers. Ninety-six samples of RNHBM were prepared and exposed for 24 h to 17%, 42.5%, or 85% glycerol, or to distilled H2 O (control). Sections were stained with haematoxylin and eosin (H&E) to evaluate epithelial thickness or used for immunohistochemistry to measure expression of Ki67 (proliferation), cleaved caspase-3 (apoptosis), and E-cadherin (tissue-integrity). Positive cells and cell layers, as detected by immunohistochemistry, were counted. Epithelial thickness, proliferation, and apoptosis were significantly increased by exposure to 42.5% and 85% glycerol. No significant differences in apoptosis or proliferation were found between controls and RNHBM exposed to 17% glycerol. E-cadherin expression was not significantly affected by exposure to any of the concentrations of glycerol tested. This study shows that glycerol affects tissue homeostasis, but not tissue integrity, of RNHBM at glycerol concentrations above 42.5%.

In Vivo Host Response and Degradation of Copolymer Scaffolds Functionalized with Nanodiamonds and Bone Morphogenetic Protein 2.

The aim is to evaluate the effect of modifying poly[(l-lactide)-co-(ε-caprolactone)] scaffolds (PLCL) with nanodiamonds (nDP) or with nDP+physisorbed BMP-2 (nDP+BMP-2) on in vivo host tissue response and degradation. The scaffolds are implanted subcutaneously in Balb/c mice and retrieved after 1, 8, and 27 weeks. Molecular weight analysis shows that modified scaffolds degrade faster than the unmodified. Gene analysis at week 1 shows highest expression of proinflammatory markers around nDP scaffolds; although the presence of inflammatory cells and foreign body giant cells is more prominent around the PLCL. Tissue regeneration markers are highly expressed in the nDP+BMP-2 scaffolds at week 8. A fibrous capsule is detectable by week 8, thinnest around nDP scaffolds and at week 27 thickest around PLCL scaffolds. mRNA levels of ALP, COL1α2, and ANGPT1 are significantly upregulating in the nDP+BMP-2 scaffolds at week 1 with ectopic bone seen at week 8. Even when almost 90% of the scaffold is degraded at week 27, nDP are observable at implantation areas without adverse effects. In conclusion, modifying PLCL scaffolds with nDP does not aggravate the host response and physisorbed BMP-2 delivery attenuates inflammation while lowering the dose of BMP-2 to a relatively safe and economical level.

Human bone marrow mesenchymal stem cells induce collagen production and tongue cancer invasion.

Tumor microenvironment (TME) is an active player in carcinogenesis and changes in its composition modify cancer growth. Carcinoma-associated fibroblasts, bone marrow-derived multipotent mesenchymal stem cells (BMMSCs), and inflammatory cells can all affect the composition of TME leading to changes in proliferation, invasion and metastasis formation of carcinoma cells. In this study, we confirmed an interaction between BMMSCs and oral tongue squamous cell carcinoma (OTSCC) cells by analyzing the invasion progression and gene expression pattern. In a 3-dimensional myoma organotypic invasion model the presence of BMMSCs inhibited the proliferation but increased the invasion of OTSCC cells. Furthermore, the signals originating from OTSCC cells up-regulated the expression of inflammatory chemokines by BMMSCs, whereas BMMSC products induced the expression of known invasion linked molecules by carcinoma cells. Particularly, after the cell-cell interactions, the chemokine CCL5 was abundantly secreted from BMMSCs and a function blocking antibody against CCL5 inhibited BMMSC enhanced cancer invasion area. However, CCL5 blocking antibody did not inhibit the depth of invasion. Additionally, after exposure to BMMSCs, the expression of type I collagen mRNA in OTSCC cells was markedly up-regulated. Interestingly, also high expression of type I collagen N-terminal propeptide (PINP) in vivo correlated with the cancer-specific mortality of OTSCC patients, whereas there was no association between cancer tissue CCL5 levels and the clinical parameters. In conclusion, our results suggest that the interaction between BMMSC and carcinoma cells induce cytokine and matrix molecule expression, of which high level of type I collagen production correlates with the prognosis of OTSCC patients.

The effects of CD44 down-regulation on stem cell properties of head and neck cancer cell lines.

BACKGROUND: There is now substantial evidence that only a subpopulation of cells in solid cancers is able to sustain tumour growth and to re-initiate new tumours. Various cancers and cancer-derived cell lines, including head and neck squamous cell carcinomas (HNSCC), have a subpopulation of cancer stem cells (CSCs), marked by high levels of expression of the CD44 adhesion molecule. However, it has been unclear whether, in addition to acting as a marker, CD44 has functions that directly influence stem cell properties. The aim of this study was to investigate the role of CD44 in the maintenance of the CSC population in HNSCC cell lines. METHODS: CD44 was down-regulated either by treating cultures with 1 mM sodium butyrate or by the more specific method of knockdown with siRNAs directed against CD44. Changes in CD44 expression levels were assessed at the mRNA and protein levels, and the effects of CD44 down-regulation on cell proliferation and on the fate of the CSC subpopulations were assessed. RESULTS: Reduced CD44 expression resulted in a decreased rate of population expansion, both initially and on repassage, and there was an alteration in colony morphologies indicative of stem cell loss. Down-regulation of CD44 also led to reduced expression of Oct4A, an alternative marker of CSCs. CONCLUSIONS: The results suggest that CD44 has a functional role in maintaining stem cell properties in HNSCC cell lines and provides support for the concept that therapies targeting CD44, or its related signalling pathways, may allow development of more efficient treatment strategies.

The use of salivary cytokines as a screening tool for oral squamous cell carcinoma : A review of the literature.

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck cancer. The 5-year survival rate has remained below 50% over the last two decades, and new tools for early diagnosis are needed. Saliva has been used for diagnosis of several systemic diseases, and its use for diagnosis of OSCC has been sought extensively. Among the many salivary analytes for diagnosis of OSCC, accumulating evidences indicate the possibility of using salivary cytokines. Overproduction of proinflammatory, proangiogenic cytokines by OSCC cells has been reported, and their role in tumor progression and angiogenesis is well established. However, many inflammatory conditions and immunological diseases could affect the levels of cytokines in serum and saliva. This article has reviewed publications in this matter, and some strengths and weaknesses have been pointed out. Conclusively, large-scale investigations are required for validation of the use of salivary cytokines for diagnosis of OSCC, with consideration to the influential role of periodontal inflammation in their levels.

Limited in-depth invasion of Fusobacterium nucleatum into in vitro reconstructed human gingiva.

OBJECTIVE: Fusobacterium nucleatum is an opportunistic pathogen with a key role in subgingival plaque formation and it is found in increased numbers in periodontally affected sites. This study aimed to investigate the potential of F. nucleatum to penetrate and induce alterations in an in vitro reconstructed human gingival mucosa model. METHODS: Three-dimensional (3D) organotypic models of human gingiva were engineered using primary gingival keratinocytes and fibroblasts. The reconstructed tissues were challenged with four different strains of fluorescently labelled F. nucleatum in suspension placed on top of epithelial layers. Confocal laser scanning was used to assess the presence of fusobacteria through the organotypic model. Apoptosis (cleaved caspase-3) and cell proliferation (Ki-67) were evaluated by the use of immunohistochemistry in 3D-tissue models. Quantitative real-time PCR was performed to investigate the mRNA expression for MMP-13 and E-cadherin in both 3D-tissues and monolayers. RESULTS: F. nucleatum invaded the superficial epithelial layers of gingival 3D-tissue models. Challenged tissues showed accentuated shedding of superficial layers and increased number of cleaved caspase-3 and Ki-67 positive cells than controls, although not statistically significant. Levels of E-cadherin and MMP-13 mRNA were not significantly perturbed in multilayer culture. A variable and disproportionate response of MMP-13 mRNA level resulted in challenged primary keratinocytes in monolayers, compared to multilayer culture. CONCLUSION: These results indicate that F. nucleatum is able to invade superficially a differentiated, stratified gingival epithelium in vitro and triggers the efficient elimination of bacterial infection through epithelial shredding without causing a permanent damage of the tissue.

Fusobacterium nucleatum enters normal human oral fibroblasts in vitro.

BACKGROUND: Fusobacterium nucleatum, a commensal opportunistic oral bacterium, is capable of invading gingival epithelial cells, but the entrance into human primary oral fibroblast cells has not been documented. This study evaluated the ability of three strains of F. nucleatum (F. nucleatum ssp. nucleatum, F. nucleatum ssp. polymorphum, and F. nucleatum ssp. vincentii) to enter gingival fibroblasts (GFs) and periodontal ligament fibroblasts (PLFs). METHODS: GFs and PLFs were cocultured for various periods of time with different strains of F. nucleatum. Scanning and transmission electron microscopy, together with confocal laser scanning microscopy, were used to visualize the entrance and presence of bacteria in host cells. Flow cytometry was performed to compare the load of internalized bacteria in GFs and PLFs exposed for 3 and 5 hours to live F. nucleatum labeled with fluorescein isothiocyanate. RESULTS: All three strains of F. nucleatum were found entering and located in the cytoplasm of GFs and PLFs after 1 hour of exposure. Flow cytometry tests revealed a significant increase in the fluorescent signal, compared to baseline, derived from bacteria internalized in fibroblasts exposed for 3 hours (P <0.001); a further increase was found at 5 hours. The greatest bacterial mass in exposed fibroblasts of both types was of F. nucleatum ssp. polymorphum; the smallest was of F. nucleatum ssp. vincentii. Although not statistically significant, PLFs had a higher bacterial load than corresponding GFs. CONCLUSION: F. nucleatum was capable of entering GFs and PLFs in a manner that is dependent on the cell type and the bacterial strain.

Epithelial stem cells and malignancy.

The renewal of normal epithelia depends on a small sub-population of cells, termed somatic stem cells, whose primary characteristic is an ability for indefinite self-renewal. Evidence is accumulating that the growth of tumours similarly depends on a sub-population of malignant stem cells, often termed tumour-initiating cells. Tumour-initiating sub-populations within solid tumours have been identified by their cell surface expression of various phenotypic markers and by their ability to regenerate tumours in immune-deficient mice. Cells with such clonogenic abilities differ consistently from the remainder of the cell population in cellular properties such as size, adhesiveness, dye exclusion, and patterns of gene expression. Sub-populations of malignant cells freshly isolated from tumours also show differing patterns of expression of molecules related to stem cell maintenance and asymmetric division. As the cells ultimately responsible for tumour renewal, malignant stem cells appear to form the necessary target of therapy but some findings indicate greater resistance of these cells to the induction of apoptotic cell death and their potential failure to respond effectively to standard therapeutic procedures. Of particular interest, cells with clonogenic properties and expression patterns similar to those of tumour-initiating cells in vivo persist in malignant cell lines and show similar apoptotic resistance. Cell lines may thus provide a model for analysis of malignant stem cell properties and may be useful for the development of appropriate methods for their elimination.

Khat (Catha edulis) induces reactive oxygen species and apoptosis in normal human oral keratinocytes and fibroblasts.

Khat chewing is widely practiced in Eastern Africa and the Middle East. Khat is genotoxic to cells within the oral mucosa, and several studies have suggested an association between khat use and oral lesions like hyperkeratosis and oral cancer. This study investigated the mechanism of khat-induced cytotoxicity using primary normal human oral keratinocytes (NOK) and fibroblasts (NOF). Khat induced rounding up of cells, plasma membrane blebbing, and condensation of nuclear chromatin within 3-6 h of exposure. The cells also showed externalization of phosphatidylserine and fragmentation of DNA. Morphological and biochemical features were compatible with cell death by apoptosis. Khat also induced an increase in cytosolic reactive oxygen species (ROS) and a depletion of intracellular glutathione (GSH) within 1 h of exposure. Antioxidants reduced ROS generation, GSH depletion and delayed the onset of cytotoxicity in both cell types. Generally, NOF cells were more sensitive to khat-induced cytotoxicity than NOK cells. These effects were elicited at concentrations of khat expected to occur in the oral cavity during khat chewing. In summary, khat induced apoptotic cell death in primary normal oral keratinocytes and fibroblasts by an early effect on mechanisms that regulate oxidative stress.

Khat induces G1-phase arrest and increased expression of stress-sensitive p53 and p16 proteins in normal human oral keratinocytes and fibroblasts.

Khat is a psychostimulant plant used by over 10 million people daily, mainly in eastern Africa and the Middle East. Previous studies have suggested an association between khat use and oral lesions such as hyperkeratosis and oral cancer. This study investigated the effects of an extract of khat on primary normal human oral keratinocytes (NOK) and normal human oral fibroblasts (NOF). Low (sublethal) concentrations of khat inhibited the proliferation of both cell types in a dose-dependent and time-dependent manner. Both NOK and NOF treated with khat accumulated in the G1-phase of the cell cycle and showed increased expression of the stress-sensitive p53 protein after 24 h. Normal human oral keratinocytes showed a profound increase in p16(INK4A) (p16) after 24 h and showed morphological changes suggesting cell differentiation. Normal human oral fibroblasts showed growth inhibition and increased expression of p21(WAF1/CIP1) (p21) within 24 h. The concentrations of khat tested in this study were within the range of those found in the oral cavity of khat chewers. The results show that stress induced by khat modulates the cell cycle in oral keratinocytes and fibroblasts. It is further speculated whether khat could have similar effects in vivo, especially in keratinocytes.