Overexpression of ALDH1 and EMT marker profile are linked with unfavorable outcome in head and neck cancer.

BACKGROUND: The aim of this research was to assess the expression of aldehyde dehydrogenase 1 (ALDH1) and epithelial-mesenchymal transition (EMT) markers in head and neck squamous cell carcinoma (HNSCC), and to correlate them with the clinical and histopathological parameters of a patient cohort with follow-up over an 8-year period. MATERIAL AND METHODS: For this, seventeen HNSCC and non-neoplastic adjacent epithelium (AE) samples were subjected to laser microdissection and real-time PCR to evaluate the mRNA expression of ALDH1, E-cadherin (E-CAD), N-cadherin (N-CAD), and vimentin (VIM). Also, immunohistochemistry was performed for ALDH1, E-CAD, N-CAD, and VIM in the tumor center (TC), invasion front (IF), and AE of the seventeen samples. Mann-Whitney, Kruskal-Wallis and Chi-square tests were used to correlate the mRNA and immunohistochemical expression with different variables, considering p<0.05. Kaplan-Meier curves were produced for local recurrence, regional metastasis and treatment. RESULTS: A mRNA overexpression of ALDH1 in primary tumors was associated with regional metastasis and a high ALDH1 immunostaining was related to metastasis and a worse patient outcome. Additionally, a favorable outcome was associated with the transition phase and an unfavorable outcome was associated with EMT event. An overall 26.9 months was observed with longer survival associated with surgery and radiotherapy. CONCLUSIONS: However, due to the intense variability inherent to the indicator proteins in the EMT process, the complete profile markers related to this biological process should be continuous investigated.

Development of an in vitro model to study tooth cystogenesis.

AIM: To describe an in vitro experimental model of cystic structure formation to conduct research on radicular cyst development. METHODOLOGY: To form spheroid structures, various numbers (1 × 104 , 5 × 104 or 1 × 105 ) of epithelial cells (HaCaT and Cal27) were seeded in 96-well plates previously coated with 1.5% low-melting agarose. After 24 h, the spheroids were collected, embedded in 3D collagen matrix and transferred to 24-well plates previously coated with polymerized collagen and kept for up to 21 days. Images of spheroids were captured at each time-point (1, 5, 9, 15 and 21 days), and samples underwent histological and confocal microscopy analyses. Spheroid area, perimeter and cell dispersion were measured. One-way Anova was used for statistical analysis. RESULTS: Both epithelial cell lines were able to generate regular and circular spheroids after 24 h of incubation regardless of cell density. Spheroid structures in the collagen matrix were uniform in most samples until day 15, when several spots that appeared to be new cultures were seen. Spheroids from HaCaT were significantly more stable than those from Cal27 (P < 0.05). Starting on the third day, the examination of histological sections revealed a cavity with epithelial lining morphology, similar to a pathological radicular cyst. CONCLUSIONS: This study describes an experimental model of cystogenesis in vitro that may be used to test theories and investigates the effects of different growth factors during cyst development and maintenance.

Non-muscle myosin II as a predictive factor in head and neck squamous cell carcinoma.

BACKGROUND: The present study attempted to provide information regarding non-muscle myosin II (MII) isoforms immunoreactivity in patients with head and neck squamous cell carcinoma (HNSCC) and analysis of the patients' clinical status after 5 years of monitoring. MATERIAL AND METHODS: A semiquantitative analysis of the immunoreactivity of the MII isoforms was performed in 54 surgical specimens and its correlation with clinical and pathological variables and prognosis was verified. Data were analyzed using chi-square, Mann-Whitney and Kruskal-Wallis tests. To evaluate the survival over the total monitoring time and any connection with the proteins studied, the Kaplan-Meier analysis was used. P values ≤0.05 were considered statistically significant. RESULTS: In the advanced stages of pathological tumor-node-metastasis, the expression of MIIB in adjacent non-neoplastic epithelial tissues tended to increase (p = 0.057). In tumoral zones there was an association of high expression among the three isoforms (MIIA/MIIB p=0,001, MIIB/MIIC p=0,006 and MIIA/MIIC p=0,012). Negative clinical evolution in patients was directly correlated to increased MIIC expression in the tumoral zone of invasion in HNSCC (p = 0.017). Based on clinical evolution after the monitoring period, patients with tumors expressing MIIC had poorer prognoses (p = 0.048). CONCLUSIONS: The present study suggests that MIIB expression in non-neoplastic adjacent epithelial tissues may indicate a potential for regional metastasis and that MIIC expression in the tumoral zone of invasion is predictive of negative evolution of the disease.

Epithelial oral mucosal cells: Do they behave differently when exposed to oral carcinogens?

OBJECTIVE: To assess the level of maturation and proliferation of epithelial cells and the correlation with immunocytochemical expression of adhesion (E-cadherin) and cell differentiation (involucrin) markers. METHODS: Cytopathological samples were obtained from four groups of patients: control (CG, n=30); alcohol/tobacco (ATG, n=31), leucoplakia (LG, n=31), and squamous cell carcinoma (SCCG, n=22). Cytopathological smears were collected from all groups for AgNOR, Papanicolaou and immunocytochemical staining. RESULTS: There was an increase in anucleated cells in ATG compared to CG and in LG compared to lesion-free groups (P<.05). In addition, there was a higher rate of intermediate cells in lesion-free groups than in LG (P=.001). When these findings were correlated with positive E-cadherin expression, there was a smaller number of anucleated and intermediate cells (P<.05). The proliferation rate was higher in the SCCG than in the CG (P<.05) and in the ATG compared to LG (P<.05). Moreover, cell proliferation increased in the presence of positive E-cadherin expression in the ATG and LG. No statistically significant results were obtained for involucrin analysis. CONCLUSION: Cytopathology combined with quantitative techniques such as Papanicolaou, AgNOR, and immunocytochemical expression of E-cadherin detects changes associated with oral carcinogenesis. The innovative approach used in this study allows assessing the expression of cell adhesion (E-cadherin) and differentiation (involucrin) markers by means of oral mucosal cytopathology. The E-cadherin imunocytochemical expression indicated changes associated with the oral carcinogenesis process. An increase in cell proliferation rate in oral squamous cell carcinoma group was associated with the lower immunoexpression of E-cadherin. Cytopathology combined with quantitative techniques and immunocytochemical expression of E-cadherin may detect early alterations associated with oral carcinogenesis.

Autophagy analysis in oral carcinogenesis.

OBJECTIVE: The aim of this study was to evaluate the levels of autophagy in oral leukoplakia and squamous cell carcinoma and to correlate with clinical pathological features, as well as, the evolution of these lesions. METHODOLOGY: 7 Normal oral mucosa, 51 oral leukoplakias, and 120 oral squamous cell carcinomas (OSCC) were included in the study. Histological sections of the mucosa and leukoplakias were evaluated throughout their length, while the carcinomas were evaluated using Tissue Microarray. After the immunohistochemical technique, LC3-II positive cells were quantified in the different epithelial layers of the mucosa and leukoplakias and in the microarrays of the squamous cell carcinomas. The correlation between positive cells with the different clinical-pathological variables and with the evolution of the lesions was tested using the t test, ANOVA, and Kaplan-Meier survival analysis. RESULTS: We observed increased levels of autophagy in the oral squamous cell carcinomas (p<0.001) in relation to the other groups, but without any association with poorer evolution or survival of these patients. Among the leukoplakias, we observed a higher percentage of positive cells in the intermediate layer of the dysplastic leukoplakias (p=0.0319) and in the basal layer of lesions with poorer evolution (p=0.0133). CONCLUSION: The levels of autophagy increased during the process of oral carcinogenesis and are correlated with poorer behavior of the leukoplakias.

Nuclear changes in oral mucosa of alcoholics and crack cocaine users.

The effects of drugs of abuse on oral mucosa are only partly understood. The aims of the present study were to: (1) evaluate the frequency of nuclear changes in normal-appearing oral mucosa of alcoholics and crack cocaine users and (2) assess their association with cell proliferation rate. Oral smears were obtained from the border of the tongue and floor of the mouth of 26 crack cocaine users (24 males and 2 females), 29 alcoholics (17 males and 12 females), and 35 controls (17 males and 18 females). Histological slides were submitted to Feulgen staining to assess the frequency of micronuclei (MN), binucleated cells (BN), broken eggs (BE), and karyorrhexis (KR). A significant increase in the frequency of MN was observed in cells exfoliated from the tongue of crack cocaine users (p = 0.01), and alcoholics showed a higher frequency of KR in cells obtained from the floor of the mouth (p = 0.01). Our findings suggest that the use of crack cocaine induces clastogenic effects, whereas alcoholism is associated with higher degrees of keratinization in the floor of the mouth.

Immunoprofiling of oral squamous cell carcinomas reveals high p63 and survivin expression.

BACKGROUND: Cancer is a multifactorial disease composed of cells that show somatic mutations and epigenetic changes. The aim of this study was to investigate the expression of proteins involved in the development and maintenance of epithelia, cell cycle regulation, and apoptosis in human oral squamous cell carcinoma (OSCC) tissue samples. METHODS: A tissue microarray containing 65 primary human OSCC specimens was immunolabeled for bcl-2, survivin, epidermal growth factor receptor (EGFR), p21, p53, p63, and cleaved caspase-3. RESULTS: Samples were scored for percentage of positively stained tumor cells and staining intensity. A total immunostaining score was also calculated, using the product of percentage and intensity scores. All specimens showed high scores, > 75%, for p63 and survivin, and 75.4% of the specimens also presented high EGFR expression. All cases showed p53-positive cells. p21 showed a diffuse staining pattern. The percentage of cells positive for cleaved caspase-3 and bcl-2 was low. CONCLUSIONS: The high frequency of tumor cells expressing p63 and survivin highlights the role of these proteins in the malignant transformation of oral epithelium. Collectively, our results suggest that p63 and survivin may constitute attractive targets for cancer therapy in patients with OSCC.

Exposure to alcohol or tobacco affects the pattern of maturation in oral mucosal cells: a cytohistological study.

OBJECTIVE: To assess the maturation pattern of oral mucosal cells of patients exposed to tobacco and alcohol. METHODS: (i) Group without lesions. Smears obtained from the lower lip, border of the tongue and floor of the mouth of 31 control individuals (group I), 49 tobacco users (group II) and 27 tobacco/alcohol users (group III) were stained using the Papanicolaou method. The first 100 cells counted on each smear determined the maturation pattern and the keratinization index (KI). Analysis of variance (ANOVA) and the Tukey multiple comparison test were used for statistical analysis, at a 5% significance level. (ii) Group with lesions. Cytopathological and histopathological studies were conducted for 15 patients: eight with leucoplakia without epithelial dysplasia, two with epithelial dysplasia and five with squamous cell carcinoma. RESULTS: (i) Group without lesions. Statistical analysis revealed a smaller number of superficial cells with nuclei in all sites of the group of tobacco/alcohol users (group III) when compared to the control group (group I), and this difference was statistically significant (P<0.005). (ii) Group with lesions. The severity of histopathological findings increased with the increase in the number of cells of the deeper epithelial layers, with a statistically significant difference in the number of intermediate (P=0.013) and parabasal cells (P=0.049), which increased with the severity of the epithelial maturation disorder: leucoplakias with dysplasia had a greater number of intermediate and parabasal cells than leucoplakias without dysplasia; and the number in squamous cell carcinomas was greater than in leucoplakias with dysplasia. CONCLUSION: The maturation pattern of cells in the three anatomic sites showed changes that may be associated with the synergistic effect of tobacco and alcohol. Also, the severity of histopathological findings was associated with the increase in the number of cells in the deeper epithelial layers.

Relationship between Candida infection and immune cellular response in inflammatory hyperplasia.

OBJECTIVES: To analyze and quantify the CD8(+) and CD4(+) T-lymphocyte populations in inflammatory hyperplasia and to establish the relationship between the frequency and location of these cells and Candida infection. METHODS: Samples of inflammatory hyperplasia were stained with PAS for evidence of Candida sp. and were classified in two groups, infected and control, according to the presence or absence of infection. After immunoreaction with specific anti-CD4 and anti-CD8 monoclonal antibodies, the distribution and frequency of the positive cells were analyzed in 41 cases (19 controls without Candida sp. and 22 infected cases). Lymphocytes were quantified in the three consecutive fields where the inflammatory infiltration was concentrated. RESULTS: There was no relationship between the frequency and location of CD4(+) T cells and Candida sp. infection. The number of CD8(+) cells close to the fungi hyphae as well as the total number of CD8(+) T cells present in inflammatory hyperplasia were higher in the Candida sp. group than in the control noninfected group (P < 0.05). CONCLUSION: Since the CD8(+) T cells were distributed according to the location of Candida sp. hyphae, and since a higher CD8(+)/total lymphocytes ratio was observed in the infected group, we suggest a role for CD8(+) T cells in the defense against Candida in oral infections associated with inflammatory hyperplasia in immunocompetent individuals.