p53 expression in leukoplakia and carcinoma of the tongue

There is growing interest in assessing multistep carcinogenesis and predicting its course using different molecular markers. TP53 is a tumor suppressor gene and appears to be one of the molecular targets of tobacco-related carcinogens in oral cancer. The present study evaluated the role of p53 expression in patients with leukoplakia and carcinoma of the tongue. p53 expression was studied by immunohistochemistry. All patients with leukoplakia of the tongue were male tobacco users. Nuclear staining of p53 was observed in 79% of those patients. Fifty percent, 25% and 4% of the patients expressed 1+, 2+ and 3+ nuclear staining, respectively. When leukoplakia patients were graded according to histopathology, 67% had hyperplasia and 33% had dysplasia. Nuclear p53 accumulation was 88% in hyperplasia and 62% in dysplasia. In patients with tongue cancer, nuclear accumulation of p53 was seen in only 19% of the tumors, with a staining intensity of 1+ in 13%, 2+ in 2% and 3+ in 4% of the tumors. The prevalence of nuclear p53 positivity (79%) was significantly higher in patients with leukoplakia than in patients with tongue cancer (19%; chi2 = 34.32, r = -0.45, df = 1, p = 0.0001; odds ratio (OR) = 16.66, 95% CI, 5.25-52.86). Therefore, leukoplakia patients who show p53 expression have a higher risk of developing tongue cancer than those who do not show p53 expression. As the percentage of positivity of nuclear p53 was very low, none of the clinicopathological parameters or disease status showed any significant association with it. The interesting finding is that none of the female cancer patients showed nuclear p53 expression. Therefore, p53 accumulation is believed to be an early event in neoplastic progression of the tongue.     

Improvement in the risk assessment of oral leukoplakia through morphology-related copy number analysis

Oral leukoplakia is the most common type of oral potentially malignant disorders and considered a precursor lesion to oral squamous cell carcinoma. However, a predictor of oral leukoplakia prognosis has not yet been identified. We investigated whether copy number alteration patterns may effectively predict the prognostic outcomes of oral leukoplakia using routinely processed paraffin sections. Comparison of copy number alteration patterns between oral leukoplakia with hyperplasia(HOL,n=22) and dysplasia(DOL, n=21) showed that oral leukoplakia with dysplasia had a higher copy number alteration rate(86%)than oral leukoplakia with hyperplasia(46%). Oral leukoplakia with dysplasia exhibited a wider range of genomic variations across all chromosomes compared with oral leukoplakia with hyperplasia. We also examined a retrospective cohort of 477 patients with oral leukoplakia with hyperplasia with detailed follow-up information. The malignant transformation(MT, n=19)and leukoplakia recurrence(LR, n=253) groups had higher frequencies of aneuploidy events and copy number loss rate than the free of disease(FD, n=205) group. Together, our results revealed the association between the degree of copy number alterations and the histological grade of oral leukoplakia and demonstrated that copy number alteration may be effective for prognosis prediction in oral leukoplakia patients with hyperplasia.     

A Quest for miRNA Bio-Marker: A Track Back Approach from Gingivo Buccal Cancer to Two Different Types of Precancers

Deregulation of miRNA expression may contribute to tumorigenesis and other patho-physiology associated with cancer. Using TLDA, expression of 762 miRNAs was checked in 18 pairs of gingivo buccal cancer-adjacent control tissues. Expression of significantly deregulated miRNAs was further validated in cancer and examined in two types of precancer (leukoplakia and lichen planus) tissues by primer-specific TaqMan assays. Biological implications of these miRNAs were assessed bioinformatically. Expression of hsa-miR-1293, hsa-miR-31, hsa-miR-31* and hsa-miR-7 were significantly up-regulated and those of hsa-miR-206, hsa-miR-204 and hsa-miR-133a were significantly down-regulated in all cancer samples. Expression of only hsa-miR-31 was significantly up-regulated in leukoplakia but none in lichen planus samples. Analysis of expression heterogeneity divided 18 cancer samples into clusters of 13 and 5 samples and revealed that expression of 30 miRNAs (including the above-mentioned 7 miRNAs), was significantly deregulated in the cluster of 13 samples. From database mining and pathway analysis it was observed that these miRNAs can significantly target many of the genes present in different cancer related pathways such as “proteoglycans in cancer”, PI3K-AKT etc. which play important roles in expression of different molecular features of cancer. Expression of hsa-miR-31 was significantly up-regulated in both cancer and leukoplakia tissues and, thus, may be one of the molecular markers of leukoplakia which may progress to gingivo-buccal cancer.     

The use of CA-IX as a diagnostic method for oral leukoplakia

Abstract

The presence and degree of dysplasia are important diagnostic and prognostic criteria for oral leukoplakia, but evaluation of dysplasia is difficult and subjective. Carbonic anhydrase-IX (CA-IX) is expressed primarily in tumor cells and is considered a specific hypoxia marker. We investigated the role of CA-IX in oral leukoplakia. We investigated 30 specimens of oral leukoplakia and 35 dysplasia specimens adjacent to the tumor margin. We analyzed clinical variables including age, sex, degree of dysplasia, and smoking, clinical appearance of leukoplakia, number of lesions, location, size, clinical monitoring, malignant transformation and recurrence. For the immunohistochemical study, we used a noncommercial monoclonal antibody against human CA-IX MAb M75. We found greater CA-IX positivity in nonsmokers, erythroplakia and mottled leukoplakia, those located on the tongue, patients with multiple lesions, 2–4 cm leukoplakias and in recurrent cases, although differences were not statistically significant. All lesions in all samples without dysplasia were negative for CA-IX; however, for all other categories of dysplasia, the percentages of positivity and negativity varied. Regarding the diagnostic index values, we found a sensitivity of 32%, specificity of 100%, a positive predictive value of 100% and a negative predictive value of 13%. Leukoplakias appear mainly in females and potentially are malignant; more than 90% have some degree of dysplasia, and therefore require close clinical and histopathological monitoring. The CA-IX immunohistochemical marker may be useful for screening samples without dysplasia owing to its high specificity.     

In situ estimation of tendon material properties: Differences between muscles of the feline hindlimb

Recent experiments to characterize the short-range stiffness (SRS)–force relationship in several cat hindlimb muscles suggested that the there are differences in the tendon elastic moduli across muscles [Cui, L., Perreault, E.J., Maas, H., Sandercock, T.G., 2008. Modeling short-range stiffness of feline lower hindlimb muscles. J. Biomech. 41 (9), 1945–1952.]. Those conclusions were inferred from whole muscle experiments and a computational model of SRS. The present study sought to directly measure tendon elasticity, the material property most relevant to SRS, during physiological loading to confirm the previous modeling results. Measurements were made from the medial gastrocnemius (MG), tibialis anterior (TA) and extensor digitorum longus (EDL) muscles during loading. For the latter, the model indicated a substantially different elastic modulus than for MG and TA. For each muscle, the stress–strain relationship of the external tendon was measured in situ during the loading phase of isometric contractions conducted at optimum length. Young's moduli were assessed at equal strain levels (1%, 2% and 3%), as well as at peak strain. The stress–strain relationship was significantly different between EDL and MG/TA, but not between MG and TA. EDL had a more apparent toe region (i.e., lower Young's modulus at 1% strain), followed by a more rapid increase in the slope of the stress–strain curve (i.e., higher Young's modulus at 2% and 3% strain). Young's modulus at peak strain also was significantly higher in EDL compared to MG/TA, whereas no significant difference was found between MG and TA. These results indicate that during natural loading, tendon Young's moduli can vary considerably across muscles. This creates challenges to estimating muscle behavior in biomechanical models for which direct measures of tendon properties are not available.     

Expression of cyclooxygenase-1 and cyclooxygenase-2 in normal and pathological human oral mucosa

Cyclooxigenase (COX) is the rate-limiting enzyme for the conversion of arachidonic acid (AA) to prostaglandins (PGs). Two isoforms of COX have been identified: COX-1 is constitutively expressed in many cells and is involved in cell homeostasis, angiogenesis and cell-cell signalling; COX-2 is not expressed in normal condition however it is strongly expressed in inflammation. The oral cavity is constantly exposed to physical and chemical trauma that could lead to mucosal reactions such as hyperplasia, dysplasia and cancer. Early diagnosis is the most important issue to address for a positive outcome of oral cancer; therefore it would be useful to identify molecular markers whose expression is associated with the various stages of oral cancer progression. Since COX enzyme has been involved, with different mechanisms, in the development and progression of malignancies we decided to investigate the expression and localization of COX-1 and COX-2 in normal human oral mucosa and three different pathologies (hyperplasia, dysplasia and carcinoma) by immunohistochemistry and RT-PCR. COX-1 mRNA and protein have been detected already in normal oral mucosa and their expression progressively increases from normal samples towards hyperplasia, dysplasia and finally carcinoma. On the contrary, COX-2 is not expressed in the normal tissue, starts to be expressed in hyperplasia, reaches the maximum activation in dysplasia and then starts to be downregulated in carcinoma.     

Morphological properties of the last primaries,the tail feathers,and the alulae of Accipiter nisus,Columba livia,Falco peregrinus,and Falco tinnunculus

We investigated the mechanical properties (Young's modulus, bending stiffness, barb separation forces) of the tenth primary of the wings, of the alulae and of the middle tail feathers of Falco peregrinus. For comparison, we also investigated the corresponding feathers in pigeons (Columba livia), kestrels (Falco tinnunculus), and sparrowhawks (Accipiter nisus). In all four species, the Young's moduli of the feathers ranged from 5.9 to 8.4 GPa. The feather shafts of F. peregrinus had the largest cross‐sections and the highest specific bending stiffness. When normalized with respect to body mass, the specific bending stiffness of primary number 10 was highest in F. tinnunculus, while that of the alula was highest in A. nisus. In comparison, the specific bending stiffness, measured at the base of the tail feathers and in dorso‐ventral bending direction, was much higher in F. peregrinus than in the other three species. This seems to correlate with the flight styles of the birds: F. tinnunculus hovers and its primaries might therefore withstand large mechanical forces. A. nisus has often to change its flight directions during hunting and perhaps needs its alulae for this maneuvers, and in F. peregrinus, the base of the tail feathers might need a high stiffness during breaking after diving. J. Morphol. 276:33–46, 2015. © 2014 Wiley Periodicals, Inc.     

Normal and Fibrotic Rat Livers Demonstrate Shear Strain Softening and Compression Stiffening: A Model for Soft Tissue Mechanics

Tissues including liver stiffen and acquire more extracellular matrix with fibrosis. The relationship between matrix content and stiffness, however, is non-linear, and stiffness is only one component of tissue mechanics. The mechanical response of tissues such as liver to physiological stresses is not well described, and models of tissue mechanics are limited. To better understand the mechanics of the normal and fibrotic rat liver, we carried out a series of studies using parallel plate rheometry, measuring the response to compressive, extensional, and shear strains. We found that the shear storage and loss moduli G’ and G” and the apparent Young''s moduli measured by uniaxial strain orthogonal to the shear direction increased markedly with both progressive fibrosis and increasing compression, that livers shear strain softened, and that significant increases in shear modulus with compressional stress occurred within a range consistent with increased sinusoidal pressures in liver disease. Proteoglycan content and integrin-matrix interactions were significant determinants of liver mechanics, particularly in compression. We propose a new non-linear constitutive model of the liver. A key feature of this model is that, while it assumes overall liver incompressibility, it takes into account water flow and solid phase compressibility. In sum, we report a detailed study of non-linear liver mechanics under physiological strains in the normal state, early fibrosis, and late fibrosis. We propose a constitutive model that captures compression stiffening, tension softening, and shear softening, and can be understood in terms of the cellular and matrix components of the liver.     

Immunoexpression of tenascin as a predictor of the malignancy potential of oral leukoplakia associated with a tobacco habit

Oral leukoplakia is a morphological alteration of tissue that is an early indicator for malignancy. Tenascin (TN) is a large hexameric extracellular matrix (ECM) protein with anti-adhesive properties that fosters cell migration during development, wound healing and tissue remodeling; it is present in small amounts in adult tissues. Overexpression of TN in a pathological condition may be either a cause or a consequence of the disease. We evaluated the efficacy of TN for early prediction of tobacco-associated oral cancers. We studied retrospectively 95 cases of oral leukoplakia, including mild, moderate and severe cases, using immunohistochemistry for TN. We evaluated the intensity, area and pattern of TN expression. Greater intensity and area of TN expression was observed in mild and severe dysplasia than in moderate dysplasia. Most cases showed a reticular pattern of expression, especially in mild and moderate dysplasia; a fibrillar pattern was more evident in severe dysplasia. We also observed homogeneous expression pattern in some cases. TN is a marker for dysplastic changes in epithelium and its expression may be helpful for predicting the malignancy potential of tobacco-associated oral leukoplakia.     

Micronucleus frequencies in exfoliated buccal cells in normal mucosa, precancerous lesions and squamous cell carcinoma

OBJECTIVE: To assess the value of micronuclei in the characterization of precancerous lesions of the oral cavity with reference to their likelihood of progressing to malignant lesions. STUDY DESIGN: The frequency of micronuclei was determined in exfoliated cells from normal oral mucosa, a preneoplastic condition (leukoplakia) and precancerous lesions with and without dysplasia, squamous cell carcinomas and sites of previous carcinomas that had been removed. RESULTS: Average micronucleus frequencies were increased in precancerous lesions as compared to normal mucosa and further increased in carcinomas, suggesting that micronuclei are a biomarker of neoplastic progression in this type of cancer. With all samples, micronucleus frequencies were systematically higher when cells were collected by vigorous than by light scraping, suggesting a decreasing gradient from basal to superficial layers of mucosa. The micronucleus frequency did not vary with the sex or age of patients, while it did vary with the anatomic site of the lesions. CONCLUSION: Although the gradual increase in micronucleus counts from normal mucosa to precancerous lesions to carcinomas suggests a link of this biomarker with neoplastic progression, the large overlapping of data prevents its use as a predictor of progression of precancerous lesions to malignancy in individual patients.     

Simulation of muscle and adipose tissue deformation in the passive human pharynx

Quantifying the contribution of passive mechanical deformation in the human pharynx to upper airway collapse is fundamental to understanding the competing biomechanical processes that maintain airway patency. This study uses finite element analysis to examine deformation in the passive human pharynx using an intricate 3D anatomical model based on computed tomography scan images. Linear elastic properties are assigned to bone, cartilage, ligament, tendon, and membrane structures based on a survey of values reported in the literature. Velopharyngeal and oropharyngeal cross-sectional area versus airway pressure slopes are determined as functions of Young's moduli of muscle and adipose tissue. In vivo pharyngeal mechanics for small deformations near atmospheric pressure are matched by altering Young's moduli of muscle and adipose tissue. The results indicate that Young's moduli ranging from 0.33 to 14 kPa for muscle and adipose tissue matched the in vivo range of area versus pressure slopes. The developed anatomical model and determined Young's moduli range are expected to be useful as a starting point for more complex simulations of human upper airway collapse and obstructive sleep apnea therapy.     

Immunohistochemical detection of early-stage carcinogenesis of oral leukoplakia by increased DNA-instability and various malignancy markers

The degree of DNA instability as determined by immunohistochemical staining with anti-single-stranded DNA antibody after acid hydrolysis (the DNA instability test) was used as a marker of malignancy. The test was applied to tissues of oral leukoplakia assessed histopathologically as hyperplasia (38 cases), mild (12 cases), moderate (11 cases) and severe (8 cases) dysplasia, and invasive squamous cell carcinoma (SCC, 20 cases). Tissues were subjected to immunohistochemical staining for proliferating cell nuclear antigen (PCNA), p53, DNA-fragmentation factor 45 (DFF45), analysis of various AgNORs parameters, and triple immunostaining for vascular endothelial growth factor (VEGF), CD34, and PCNA. The DNA instability test was positive in 20 (100%) SCC cases, 8 (100%) severe dysplasia cases, 8 (72.7%) moderate dysplasia cases, 6 (50.0%) mild dysplasia cases, and 9 (23.7%) hyperplasia cases, indicating malignancy. The proportion of lesions positive for PCNA, p53, DFF45, and values of AgNORs parameters steadily increased from hyperplasia to mild, moderate and severe dysplasia, and SCC, especially in those showing positive DNA instability test, indicative of malignancy. Based on these results, 44.9% of leukoplakia were malignant tissues, namely carcinoma in situ. The proportion of PCNA-positive vascular endothelial cells in the vicinity of VEGF-positive epithelial lesion was significantly higher than that of negative DNA instability lesions, as revealed by immunohistochemical triple staining for VEGF, CD34, and PCNA. Our results suggest that increased DNA instability, enhanced proliferative activity, p53 mutation, and induction of DFF45 and VEGF may allow cancer cell proliferation, enhance their survival by escaping apoptosis, and provide abundant nutrients during early-stage carcinogenesis of oral leukoplakia.     

The Control of Precancerous Conditions of the Colon and Rectum

The early diagnosis and treatment of precancerous lesions is an important means of control of intestinal cancer. The precancerous tendency is relatively slight with solitary colo-rectal polyps, ulcerative colitis and bilharzial infestation, but it is exceptionally severe in familial colonic polyposis.For more than 30 years, research into the inheritance and treatment of familial polyposis has been continued at St. Mark''s Hospital, London, England, and more than 90 families have been investigated and family pedigrees prepared. These now include 1050 “members”, of whom 343 have had either polyposis or intestinal cancer, or both, the number with cancer being 243. One hundred and one of these 343 patients have been operated on at St. Mark''s, 61 undergoing colectomy and ileorectal anastomosis.Cancer control is achieved by keeping under supervision all members of a family who have already developed polyposis or who may do so later.     

Spatial Mapping of the Biomechanical Properties of the Pericellular Matrix of Articular Cartilage Measured In Situ via Atomic Force Microscopy

In articular cartilage, chondrocytes are surrounded by a narrow region called the pericellular matrix (PCM), which is biochemically, structurally, and mechanically distinct from the bulk extracellular matrix (ECM). Although multiple techniques have been used to measure the mechanical properties of the PCM using isolated chondrons (the PCM with enclosed cells), few studies have measured the biomechanical properties of the PCM in situ. The objective of this study was to quantify the in situ mechanical properties of the PCM and ECM of human, porcine, and murine articular cartilage using atomic force microscopy (AFM). Microscale elastic moduli were quantitatively measured for a region of interest using stiffness mapping, or force-volume mapping, via AFM. This technique was first validated by means of elastomeric models (polyacrylamide or polydimethylsiloxane) of a soft inclusion surrounded by a stiff medium. The elastic properties of the PCM were evaluated for regions surrounding cell voids in the middle/deep zone of sectioned articular cartilage samples. ECM elastic properties were evaluated in regions visually devoid of PCM. Stiffness mapping successfully depicted the spatial arrangement of moduli in both model and cartilage surfaces. The modulus of the PCM was significantly lower than that of the ECM in human, porcine, and murine articular cartilage, with a ratio of PCM to ECM properties of ∼0.35 for all species. These findings are consistent with previous studies of mechanically isolated chondrons, and suggest that stiffness mapping via AFM can provide a means of determining microscale inhomogeneities in the mechanical properties of articular cartilage in situ.     

Reprogramming cardiomyocyte mechanosensing by crosstalk between integrins and hyaluronic acid receptors

The elastic modulus of bioengineered materials has a strong influence on the phenotype of many cells including cardiomyocytes. On polyacrylamide (PAA) gels that are laminated with ligands for integrins, cardiac myocytes develop well organized sarcomeres only when cultured on substrates with elastic moduli in the range 10 kPa-30 kPa, near those of the healthy tissue. On stiffer substrates (>60 kPa) approximating the damaged heart, myocytes form stress fiber-like filament bundles but lack organized sarcomeres or an elongated shape. On soft (<1 kPa) PAA gels myocytes exhibit disorganized actin networks and sarcomeres. However, when the polyacrylamide matrix is replaced by hyaluronic acid (HA) as the gel network to which integrin ligands are attached, robust development of functional neonatal rat ventricular myocytes occurs on gels with elastic moduli of 200 Pa, a stiffness far below that of the neonatal heart and on which myocytes would be amorphous and dysfunctional when cultured on polyacrylamide-based gels. The HA matrix by itself is not adhesive for myocytes, and the myocyte phenotype depends on the type of integrin ligand that is incorporated within the HA gel, with fibronectin, gelatin, or fibrinogen being more effective than collagen I. These results show that HA alters the integrin-dependent stiffness response of cells in vitro and suggests that expression of HA within the extracellular matrix (ECM) in vivo might similarly alter the response of cells that bind the ECM through integrins. The integration of HA with integrin-specific ECM signaling proteins provides a rationale for engineering a new class of soft hybrid hydrogels that can be used in therapeutic strategies to reverse the remodeling of the injured myocardium.     

Preneoplastic lesions of the lung

Lung cancer is the leading cause of cancer deaths worldwide. If we can define and detect preneoplastic lesions, we might have a chance of improving survival. The World Health Organization has defined three preneoplastic lesions of the bronchial epithelium: squamous dysplasia/carcinoma in situ; atypical adenomatous hyperplasia; and diffuse idiopathic pulmonary neuroendocrine cell hyperplasia. These lesions are believed to progress to squamous cell carcinoma, adenocarcinoma and carcinoid tumors, respectively. In this review we summarize the data supporting the preneoplastic nature of these lesions, and delve into some of the genetic changes found in atypical adenomatous hyperplasia and squamous dysplasia/carcinoma in situ.     

Cell stiffness determined by atomic force microscopy and its correlation with cell motility

BackgroundCell stiffness is a crucial mechanical property that is closely related to cell motility. AFM is the most prevalent method used to determine cell stiffness by the quantitative parameter designated as Young's modulus. Young's modulus is regarded as a biomarker of cell motility, especially in estimating the metastasis of cancer cells, because in recent years, it has been repeatedly shown that cancerous cells are softer than their benign counterparts. However, some conflicting evidence has shown that cells with higher motility are sometimes stiffer than their counterparts. Thus, the correlation between cell stiffness and motility remains a matter of debate.Scope of reviewIn this review, we first summarize the reports on correlations between cell motility and stiffness determined by AFM and then discuss the major determinants of AFM-determined cell stiffness with a focus on the cytoskeleton, nuclear stiffness and methodological issues. Last, we propose a possible correlation between cell stiffness and motility and the possible explanations for the conflicting evidence.Major conclusionsThe AFM-determined Young's modulus is greatly affected by the characteristics of the cytoskeleton, as well as the procedures and parameters used in detection. Young's modulus is a reliable biomarker for the characterization of metastasis; however, reliability is questioned in the evaluation of pharmacologically or genetically modified motility.General significanceThis review provides an overview of the current understanding of the correlation between AFM-determined cell stiffness and motility, the determinants of this detecting method, as well as clues to optimize detecting parameters.     

Mechanical properties of fibroblasts depend on level of cancer transformation

Recently, it was revealed that tumor cells are significantly softer than normal cells. Although this phenomenon is well known, it is connected with many questions which are still unanswered. Among these questions are the molecular mechanisms which cause the change in stiffness and the correlation between cell mechanical properties and their metastatic potential. We studied mechanical properties of cells with different levels of cancer transformation. Transformed cells in three systems with different transformation types (monooncogenic N-RAS, viral and cells of tumor origin) were characterized according to their morphology, actin cytoskeleton and focal adhesion organization. Transformation led to reduction of cell spreading and thus decreasing the cell area, disorganization of actin cytoskeleton, lack of actin stress fibers and decline in the number and size of focal adhesions. These alterations manifested in a varying degree depending on type of transformation. Force spectroscopy by atomic force microscopy with spherical probes was carried out to measure the Young's modulus of cells. In all cases the Young's moduli were fitted well by log-normal distribution. All the transformed cell lines were found to be 40–80% softer than the corresponding normal ones. For the cell system with a low level of transformation the difference in stiffness was less pronounced than for the two other systems. This suggests that cell mechanical properties change upon transformation, and acquisition of invasive capabilities is accompanied by significant softening.     

Influence of asymmetric stiffness on the structural and aerodynamic response of synthetic vocal fold models

The influence of asymmetric vocal fold stiffness on voice production was evaluated using life-sized, self-oscillating vocal fold models with an idealized geometry based on the human vocal folds. The models were fabricated using flexible, materially-linear silicone compounds with Young's modulus values comparable to that of vocal fold tissue. The models included a two-layer design to simulate the vocal fold layered structure. The respective Young's moduli of elasticity of the “left” and “right” vocal fold models were varied to create asymmetric conditions. High-speed videokymography was used to measure maximum vocal fold excursion, vibration frequency, and left–right phase shift, all of which were significantly influenced by asymmetry. Onset pressure, a measure of vocal effort, increased with asymmetry. Particle image velocimetry (PIV) analysis showed significantly greater skewing of the glottal jet in the direction of the stiffer vocal fold model. Potential applications to various clinical conditions are mentioned, and suggestions for future related studies are presented.