Immunoexpression of C4 Binding Protein in Oral Leukoplakia and Oral Squamous Cell Carcinoma.

BACKGROUND: The immune evasion of dysplastic cells plays an important role in suppressing the immune response and progression of malignancy. The role of the complement inhibitors in the development of oral epithelial dysplastic lesions and squamous cell carcinoma (SCC) is still unclear. OBJECTIVE: This study aimed to assess the expression of C4 binding protein (C4BP) as a complement inhibitor in oral squamous cell carcinoma and leukoplakia. METHODS: In this study, 94 samples were classified into four groups: leukoplakia with mild to moderate dysplasia, leukoplakia with severe dysplasia or carcinoma in situ, early invasive SCC, and invasive SCC. The expression of C4BP marker was evaluated by immunohistochemistry (IHC) and real-time PCR. The results were analyzed by the Kruskal-Wallis, Bonferroni adjusted Dunn's multiple comparison, and one-way ANOVA tests. RESULTS: The results of IHC revealed the expression patterns of C4BP in oral dysplasia and SCC, and indicated that the C4BP expression was not significantly different between different histopathological grades in epithelial cells and vessels (P=0.157 and P=0.123, respectively) but, it was significantly different in fibroblasts and lymphocytes (P=0.017 and P=0.043, respectively). The real-time PCR showed a significant correlation between the dysplasia grade and expression of C4BP (P<0.05). CONCLUSION: According to the results, C4BP is expressed in the cancerous tissue by the tumor cells and their surrounding stroma. In addition, upregulation of the C4BP gene as an inhibitor of the complement system is a possible strategy adopted by the tumor cells to evade the immune system.

PD-1 blockade prevents the progression of oral carcinogenesis.

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies in the head and neck with a poor prognosis. Oral cancer development is a multistep process involving carcinogenesis. Though significant advances in cancer immunotherapy over the years, there is lack of evidence for T-cell exhaustion during oral carcinogenesis. Clinical specimens from healthy donors and patients diagnosed with oral leukoplakia (OLK) or OSCC were collected for immunohistochemical staining with PD-L1, CD86, CD8, PD-1 and CTLA-4 antibodies. Meanwhile, chemically induced mouse models of oral carcinogenesis were constructed with 4-nitroquinolone-N-oxide induction. Exhaustion status of T cells was measured by flow cytometry for spleens and by multiplex immunohistochemistry for formalin-fixed paraffin-embedded lesions in multiple stages of oral carcinogenesis. The efficacy of PD-1 blockade with or without cisplatin treatment was evaluated on the mice in precancerous and OSCC stages. We observed higher expression of PD-1 in the human OLK and OSCC tissues compared with the normal, while low expression CTLA-4 in all oral mucosa tissues. Animal experiments showed that the exhausted CD4+ T cells existed much earlier than exhausted CD8+ T cells, and an increased ratio of stem-like exhausted T cells and partially exhausted T cells were detected in the experimental groups. Besides, the expression of immune checkpoint markers (PDCD1, CTLA4 and HAVCR2) was strongly positively correlated with cytokines (IFNG and IL-2). In summary, T-cell exhaustion plays a vital role in oral carcinogenesis, and PD-1 blockade can prevent the progression of oral carcinogenesis.

Leukoplakia and Immunology: New Chemoprevention Landscapes?

Oral potentially malignant disorders (OPMDs) comprise a range of clinical-pathological alterations frequently characterized by an architectural and cytological derangements upon histological analysis. Among them, oral leukoplakia is the most common type of these disorders. This work aims to analyze the possible use of drugs such as immunochemopreventive agents for OPMDs. Chemoprevention is the use of synthetic or natural compounds for the reversal, suppression, or prevention of a premalignant lesion conversion to malignant form. Experimental and in vivo data offer us the promise of molecular prevention through immunomodulation; however, currently, there is no evidence for the efficacy of these drugs in the chemoprevention action. Alternative ways to deliver drugs, combined use of molecules with complementary antitumor activities, diet influence, and better definition of individual risk factors must also be considered to reduce toxicity, improve compliance to the protocol treatment and offer a better individualized prevention. In addition, we must carefully reconsider the mode of action of many traditional cancer chemoprevention agents on the immune system, such as enhancing immunosurveillance and reversing the immune evasion. Several studies emphasize the concept of green chemoprevention as an alternative approach to accent healthy lifestyle changes in order to decrease the incidence of HNSCC.

Histo-Blood Group Antigens in Oral Cancer and Potentially Malignant Disorders.

BACKGROUND: Early detection of oral cancer is of critical importance because survival rates markedly improve when oral lesions are identified at an early stage. Aim of the present study is to investigate the expression of ABO (H) antigens in tissue specimens of oral cancer and potentially malignant disorders and to determine the role of ABO (H) antigens in tumour staging. MATERIALS AND METHODS: A prospective study was conducted on 60 cases of oral cancer and potentially malignant diseases. Specific red cell adherence test (SRCA-test) was used for studying A, B and O (H) antigens in tissue specimens and iso-antigenicity of epithelium was graded according to degree of adherence of indicator red blood cells. RESULTS: Among OSMF group, grade II adherence was seen in 53.3% cases, grade III in 33.3% cases, grade IV in 13.3% cases. In leukoplakia group, grade II adherence was seen in 26.7% cases, grade III adherence in 53.3% cases, grade IV adherence in 20% cases. Within the leukoplakia group, cases with dysplasia showed decreased adherence, compared with cases without dysplasia. Oral cancer group, negative adherence was seen in 13.3% cases, grade I adherence in 46.7% cases, grade II in 40% cases. In oral cancer group, antigen reactivity was less in poorly and moderately differentiated carcinoma, compared to well differentiated carcinoma. CONCLUSIONS: Antigen adherence and degree of loss of ABO (H) antigens in tissue specimens can be used for staging of the tumour.

Gastric parietal cell and thyroid autoantibodies in oral precancer patients.

BACKGROUND/PURPOSE: Gastric parietal cell antibody (GPCA), thyroglobulin antibody (TGA), and thyroid microsomal antibody (TMA) may be present in oral mucosal disease patients. This study mainly assessed the frequencies of serum GPCA, TGA, and TMA positivities in 131 oral precancer patients. METHODS: Serum GPCA, TGA, and TMA levels were measured in 131 oral precancer patients including 96 oral leukoplakia, 26 oral erythroleukoplakia, and 9 oral verrucous hyperplasia patients and in 131 age- and sex-matched healthy control subjects. RESULTS: We found that 131 oral precancer patients had higher frequencies of serum GPCA (10.7% vs. 2.3%, P = 0.012, statistically significant), TGA (4.6% vs. 2.3%, P = 0.498), and TMA (8.4% vs. 2.3%, P = 0.054, marginal significance) positivities than 131 healthy control subjects. We also found that 1 (0.8%), 6 (4.6%), and 16 (12.2%) oral precancer patients had the presence of three (GPCA + TGA + TMA), two (GPCA + TGA, GPCA + TMA, or TGA + TMA), or one (GPCA only, TGA only, or TMA only) autoantibody in their sera, respectively. Of 10 TGA/TMA-positive oral precancer patients whose serum thyroid-stimulating hormone (TSH) levels were measured, 80%, 10%, and 10% of these 10 TGA/TMA-positive oral precancer patients had normal, lower, and higher serum TSH levels, respectively. We also found a significantly higher GPCA positive rate in 26 smokers consuming >20 cigarettes per day than in 61 smokers consuming ≤20 cigarettes per day (P = 0.008). CONCLUSION: Approximately 17.6% of 131 oral precancer patients have serum GPCA/TGA/TMA positivity. Only approximately 20% of TGA/TMA-positive oral precancer patients have either hypothyroidism or hyperthyroidism.

MAGE-A expression in oral and laryngeal leukoplakia predicts malignant transformation.

Leukoplakia is a potential precursor of oral as well as laryngeal squamous cell carcinoma. Risk assessment of malignant transformation based on the grade of dysplasia of leukoplakia often does not lead to reliable results. However, oral squamous cell carcinoma, laryngeal squamous cell carcinoma, and leukoplakia express single or multiple members of the melanoma-associated antigens A (MAGE-A) family, while MAGE-A are absent in healthy mucosal tissue. The present study aimed at determining if there is an association between the expression of MAGE-A in leukoplakia and malignant transformation to oral or laryngeal squamous cell carcinoma. Paraffin-embedded tissues of 205 oral and laryngeal leukoplakia, 90 corresponding tumors, and 40 healthy oral mucosal samples were included in the study. The grade of dysplasia of the leukoplakia samples was determined histopathologically. The leukoplakia samples were divided into lesions that transformed to oral and laryngeal squamous cell carcinoma (n = 91) and lesions that did not (n = 114) during a 5 years follow-up. The expression of MAGE-A3/6 and MAGE-A4 was analyzed by real-time RT-PCR. The expression of MAGE-A 1-4, 6, and 12 was determined by immunohistochemistry. A total of 59.3% of the transforming leukoplakia expressed at least one of the examined antigens as opposed to an expression rate of 3.5% of all non-transforming leukoplakia. There was no MAGE-A expression in healthy oral mucosa. The risk of malignant transformation was statistically significantly associated with MAGE-A expression in immunohistochemistry (p < 0.001) and real-time RT-PCR (MAGE-A3/6, p = 0.001; MAGE-A4, p = 0.002) analyses. There was no significant association between MAGE-A expression and the grade of dysplasia ("low-grade", D0/D1; "high-grade", D2/D3) in immunohistochemistry (p = 0.412) and real-time RT-PCR (MAGE-A3/6, p = 0.667; MAGE-A4, p = 0.756). It seems that the analysis of the MAGE-A expression profile may support the identification of leukoplakia at risk for malignant transformation. Therefore, efforts should be made to establish this analysis as a routine procedure in addition to conventional histopathology.

Expression of programmed cell death-ligand 1 in oral squamous cell carcinoma and oral leukoplakia is associated with disease progress and CD8+ tumor-infiltrating lymphocytes.

OBJECTIVE: In recent years, monoclonal antibodies targeting programmed cell death-ligand 1 (PD-L1) have become a promising cancer immunotherapy. However, the role of PD-L1 in oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMDs), including oral leukoplakia (OLK), remains controversial. The aim of the present study was to investigate the expression level of PD-L1 in OSCC and OPMDs, and examine its relationship with CD8 expression and different clinicopathological features. METHOD: Expression of PD-L1 and CD8 were conducted in 41 OSCC, 21 OLK, and 25 normal mucosa samples by immunohistochemistry. Then, the density of PD-L1 expression was measured, and its correlation with CD8 expression and different clinicopathological features was analyzed. RESULTS: PD-L1 protein was detected in 97.6% of OSCC, 61.9% of OLK, and 0% of normal tissues. PD-L1 was highly expressed in human OSCC tissue (P < 0.0001), when compared to both OLK and control tissues. PD-L1 positivity was significantly associated with CD8 density (P < 0.0001, r = 0.8491). The PD-L1 high expression OSCC group displayed a trend for improved overall survival (OS) and disease-free survival (DFS) compared to the low expression group, although the differences were not significant. Moreover, the expression level of PD-L1 in OSCC was positively correlated with the pathological grade (P < 0.0001), but it was independent of age, gender, smoking, drinking, tumor size, lymph node status, or recurrence (P > 0.05). Also, there was a significant upregulation of PD-L1 expression observed in the OLK group compared to the control group (P < 0.0001). PD-L1 positivity in OLK patients was associated with gender and smoking habits (P < 0.05), but it did not correlate with age, drinking, or dysplasia (P > 0.05). CONCLUSION: The upregulation of PD-L1 may be associated with disease progress and CD8+ tumor-infiltrating lymphocytes in oral premalignant and malignant lesions.

CD163+ macrophages infiltration correlates with the immunosuppressive cytokine interleukin 10 expression in tongue leukoplakia.

Objective: Accumulating evidence suggests that macrophages are involved in the immunoediting of oral squamous cell carcinoma but the role of macrophages in oral carcinogenesis is unclear. We aimed to clarify the role of macrophages in oral leukoplakia, which is the most common oral potentially malignant disorder from immunotolerance viewpoint. Materials and methods: The study included 24 patients who underwent surgical resection for tongue leukoplakia. The relationships between macrophage markers and clinicopathological factors were assessed. Conditioned medium was harvested from the CD163+ human monocytic leukaemia cell line, THP-1. The phenotypic alteration of human oral keratinocytes by the conditioned medium treatment was assessed using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Moreover, the clinical samples were evaluated using immunohistochemistry. Results: Tongue leukoplakia tissues with high CD163+ macrophage infiltration were associated with significantly higher degrees of epithelial dysplasia, abnormal Ki-67 expression and cytokeratin13 loss when compared with the tissues with low CD163+ macrophage infiltration. In vitro, CD163+ THP-1 conditioned medium induced immunosuppressive molecules, especially interleukin-10 (IL-10) in human oral keratinocytes. The IL-10 expression levels showed significant positive correlations with not only the numbers of FOXP3+ regulatory T cells but also that of CD163+ macrophages. Conclusions: In tongue leukoplakia, CD163+ macrophages infiltration correlates with immunosuppressive cytokine IL-10 expression.

CD8+ and CD163+ infiltrating cells and PD-L1 immunoexpression in oral leukoplakia and oral carcinoma.

Overexpression of inhibitory checkpoint PD1/PD-L1 plays an important role in carcinogenesis and patients prognosis. 70 cases of oral squamous cell carcinoma (OSCC), 23 cases of oral leukoplakia (OLK), and 19 control cases were immunohistochemically stained with anti-PD-L1, -CD8, and -CD163 antibodies. PD-L1 was expressed on dysplastic and subepithelial infiltrating cells of OLK as well as on cancer and tumor-infiltrating cells of OSCC. In OSCC, PD-L1 immunoexpression was significantly increased in comparison to OLK, and control groups. The correlative study showed significant correlations between the immunoexpression of PD-L1 and the number of CD8+, CD163+ cells in both OLK and OSCC groups. We found also significant negative correlation between the number of PD-L1+ infiltrating cells and the number of CD8+ cells in OSCC, and positive correlation between the number of PD-L1+ infiltrating cells and CD163+ cells in OLK and OSCC groups. In conclusion, our study indicate that CD163+ and CD8+ infiltrating cells influence the early and subsequent stages of oral carcinogenesis. We demonstrated also that studied tumors may evade the host immune system by PD-L1 immunoexpression not only on epithelial cells but on infiltrating cells as well.

Mature dendritic cell density is affected by smoking habit, lesion size, and epithelial dysplasia in oral leukoplakia samples.

OBJECTIVE: To compare the densities of CD1a + immature and CD83+ mature dendritic cells, and inflammatory infiltrate cells between smokers and non-smokers with oral leukoplakia. Parameters associated with malignant transformation were also evaluated. DESIGN: 21 smokers and 23 non-smokers diagnosed with oral leukoplakia were obtained. Densities of inflammatory infiltrate cells were calculated in H&E sections. Immunohistochemistry using anti-CD1a and anti-CD83 was performed and densities were calculated. Comparisons and statistical analyses were performed among the groups and parameters as gender, lesion size, site, and presence of cell dysplasia were analyzed. RESULTS: A lower density of CD83+ cells was observed in smokers compared to non-smokers (P < 0.05). For samples of smokers, a lower density of CD1a + cells, CD83+ cells, and inflammatory infiltrate cells was observed in samples with <10 mm compared to samples ≥10 mm of diameter (P < 0.05), and a lower density of CD83+ cells was also observed between samples without dysplasia compared to samples with dysplasia (P < 0.05). CONCLUSION: In oral leukoplakia samples, dendritic cell density decreases in the presence of smoking habit, and increases in larger lesions and with epithelial dysplasia. Smoking habit is an external factor that contribute to alteration of the anti-tumoral immune defense system in lesions of oral leucoplakia, reinforcing that smoking elimination is important to control the development of this disease.

Potentially Malignant Oral Disorders and Cancer Transformation.

Cancer in the oral cavity is often preceded by precursor lesions. Nine oral mucosal disorders are known to have an increased risk of malignant transformation. The etiology varies from disorders caused by exogenous factors such as tobacco and autoimmune inflammation to idiopathic or inherited genetic aberrations. In this review, these potentially malignant disorders (PMDs) are described regarding clinical presentation and histopathological architecture. Special attention is paid to the underlying etiologies of PMDs and the potential pathways leading to cancer. The clinical perspective focuses on the importance of accurate and timely diagnosis.

T cells are involved in the induction of macrophage phenotypes in oral leukoplakia and squamous cell carcinoma-a preliminary report.

BACKGROUND: The prognosis of human malignancies has been shown to depend on immunological parameters, such as macrophage polarization (M1 and M2). In this study, we identify the phenotype of macrophages, and investigate an involvement of infiltrated T cells that participate in the polarization of macrophages, in oral leukoplakia (OLK), and oral squamous cell carcinoma (OSCC). METHODS: Immunohistochemical method was used to examine the number of CD68+ , CD163+ (M2), iNOS+ (M1) macrophages, and CD4+ , CD8+ , CCR4+ (Th2), CCR5+ (Th1) cells in 102 cases of OSCC: without metastases-OSCC M(-) (n = 54), and with metastases-OSCC M(+) (n = 48), 23 cases of OLK, and 18 control cases. RESULTS: The mean number of CD68+ , CD163+ , iNOS+ , CD4+ , CCR4+ , CCR5+ cells was significantly increased in OSCC M(+) group compared with OLK, OSCC M(-) and control group. We found positive correlations between the number of CD4+ T cells and CD163+ and iNOS+ macrophages as well as CCR4+ and CCR5+ cells in both OSCC groups. The mean number of CD8+ cells was significantly increased in OSCC M(-) and OLK compared with OSCC M(+) and control group. In OSCC M(+) and OSCC M(-) groups, a negative correlation between the number of CD8+ cells and CD163+ and iNOS+ macrophages was found. CONCLUSIONS: The number and co-localization of lymphocytes and macrophages in OLK and OSCC may indicate that infiltrating cells influence the early and subsequent stage of oral carcinogenesis.

Predictive value of the combination of SMAD4 expression and lymphocyte infiltration in malignant transformation of oral leukoplakia.

Oral leukoplakia (OL) is a common, potentially malignant disorder of the oral cavity. SMAD4 was initially identified as a tumor suppressor and central mediator of transforming growth factor (TGF)-ß signaling. In this study, we aimed to determine the expression patterns of SMAD4 in OL, its relationship with the degree of inflammation, and its clinical implications as a biomarker for OL malignant transformation. A total of 150 patients with OL were enrolled in this study. Paraffin-embedded sections obtained from biopsy or resection specimens were subjected to immunohistochemical analysis. Associations among the status of epithelial SMAD4 expression, stromal lymphocyte infiltration, and malignant transformation of OL were examined. Malignant transformation was significantly associated with the status of SMAD4 expression (P = 0.0017) and lymphocyte infiltration status (P = 0.0054). Cox regression analysis, based on the event-free survival (EFS), revealed that a low SMAD4 expression was a significant prognostic factor in OL patients (hazard ratio, 2.632; P = 0.043). In addition, a low SMAD4 expression was closely correlated with high lymphocyte infiltration (P = 0.00035), resulting in a significant correlation between the combination of low SMAD4 expression and high lymphocyte infiltration with malignant transformation of OL (P = 0.00027). The combination of the status of epithelial SMAD4 expression and stromal lymphocyte infiltration may be a useful biomarker for predicting malignant transformation in OL patients. These results suggest that not only epithelial SMAD4 loss, but also stromal features, may regulate the risk of malignant transformation of OL.

Expresión de E-cadherina y factor de crecimiento epidérmico en leucoplasias orales / Expression of E-cadherin and epidermal growth factor in oral leukoplakia

Introducción: se define a las leucoplasias orales como una placa blancaque no puede desprenderse por raspado y que no puede clasifi carse comoninguna otra lesión. Son lesiones con potencial maligno, relacionadascon la presencia de displasia epitelial. Estos cambios preneoplásicospueden ser evidenciados histológicamente como también a travésde técnicas que pongan en evidencia los diferentes cambios a nivelmolecular. La E-cadherina es una glicoproteína membranosa quedesempeña papeles importantes en el mantenimiento de la adhesióncélula-célula, la preservación de la polaridad del tejido epitelial y laintegridad estructural. Los factores de crecimiento epidérmico son unconjunto de moléculas de naturaleza proteica, biorreguladores, cuyafuncionalidad fundamental radica en el control del ciclo celular. Elobjetivo del presente trabajo es identifi car y comparar parámetros histológicosy moleculares predictores de riesgo de transformación malignaen leucoplasias orales. Material y métodos: El estudio correspondea un diseño observacional descriptivo. Se seleccionaron muestras de26 biopsias de leucoplasias orales, las cuales fueron evaluadas contécnica histológica de rutina y tinción con hematoxilina y eosina, luegosometidas a inmunomarcación con factor de crecimiento epidérmico yE-cadherina, donde se evaluó la intensidad de tinción y cambios en laexpresión de cada marcador, así como la localización en los diferentessubtipos celulares. Resultados: De las 26 leucoplasias observadas,16 mostraron histología con cambios hiperplásicos y 10 con cambiosdisplásicos leves a moderados. La expresión de E-cadherina no mostróalteraciones signifi cativas en leucoplasias sin displasia, sólo hubopérdida de expresión en aquellas leucoplasias con cambios displásicosde alto grado, en concordancia a los hallazgos histológicos...

Introduction: oral leukoplakia is defined as a white plaque thatcannot be removed by scraping and cannot be classifi ed as any otherdisease entity. They are potentially malignant lesions related to thepresence of epithelial dysplasia. These preneoplastic changes can bedetected histologically, as well as through techniques that demonstratediff erent changes at the molecular level. E-cadherin is a membraneglycoprotein that plays a major role in maintaining cell-cell adhesion,preserving structural integrity and the polarity of epithelial tissue.Epidermal growth factors are a group of bio-regulatory proteins,whose primary function is to control the cell cycle. The aim of thisstudy is to identify and compare the parameters for histological andmolecular markers for malignant transformation in oral leukoplakia.Material and methods: The study was observational and descriptive indesign. Samples were selected from 26 oral leukoplakia biopsies, whichwere routinely evaluated for histology and stained with hematoxylinand eosin, then subjected to immunostaining with epidermal growthfactor and E-cadherin, with the intensity of staining and changes inthe expression of each marker being evaluated. Results: Of the 26leukoplakia examined, 16 showed hyperplastic changes and 10 mildto moderate dysplastic changes. The expression of E-cadherin showedno signifi cant changes in non-dysplastic leukoplakia, while a lossof expression was found in only those leukoplakias with high-gradedysplastic changes, which was consistent with the histological fi ndings...

Salivary and serum interleukin-6 levels in proliferative verrucous leukoplakia.

BACKGROUND: Cytokines and chemokines have been analysed in patients with oral squamous cell carcinoma and potentially malignant disorders. We selected interleukin-6 (IL-6) because it is a multifunctional interleukin reported to be altered in potentially malignant oral disorders and in malignant lesions. To date, this has not been evaluated or tested in proliferative verrucous leukoplakia (PVL), however. OBJECTIVES: This study aimed to analyse the differences in serum and saliva IL-6 levels among patients with PVL, oral squamous cell carcinoma (OSCC) and healthy controls and to examine the relationship between salivary IL-6 levels and the extent of the verrucous area. METHODS: Using an enzyme-linked immunosorbent assay, we determined the serum and saliva IL-6 levels in three groups: 20 patients with PVL, 20 with OSCC and 20 healthy controls. RESULTS: There were significant (p < 0.01) differences in the serum and saliva IL-6 levels among the three groups and among the three grades of extent of the verrucous areas (p = 0.01). In the OSCC group, there was a significant difference in the saliva IL-6 levels between patients with and without lymph node metastasis at diagnosis (p = 0.02). CONCLUSIONS: We found that patients with OSCC had the highest salivary and serum IL-6 levels, while PVL had lower values than OSCC, but higher than the controls, and these altered levels were associated with the extent of the verrucous areas. CLINICAL RELEVANCE: Salivary and plasma IL-6 are altered in patients with PVL, with more extensive verrucous areas being associated to the highest IL-6 levels. This could be a significant tool for monitoring patients with PVL, their progression to more advances stages and their recurrences.

Presence of CD3-positive T-cells in oral premalignant leukoplakia indicates prevention of cancer transformation.

AIM: Leukoplakias (LPLs) are lesions in the oral mucosa that have a potential to transform into oral squamous cell carcinoma (OSCC). As the degree of immunosurveillance may be important for this transformation to occur, the aim of this study was to determine the presence of immune cells in LPLs with dysplasia in relation to later development of OSCC. MATERIALS AND METHODS: Biopsies from 16 patients with clinical diagnosis of LPL and histopathological diagnosis of hyperkeratosis with dysplasia were immunostained with antibodies to detect CD3(+) T cells, CD1a(+) LCs, Ki-67(+) and p53-expressing cells. Patients were divided into two groups: LPL with dysplasia that transformed into OSCC (LPL-dys) and that which did not (LPL-ca). RESULTS: Quantitative analyses showed significantly lower numbers of CD3(+) T-cells in LPL-ca than in LPL-dys. No significant differences were detected when comparing LPL-dys and LPL-ca regarding CD1a(+), p53(+) and Ki-67(+) cells. CONCLUSION: The number of CD3-expressing T-cells may be important for preventing malignant transformation of LPL.

Salivary level of interleukin-8 in oral precancer and oral squamous cell carcinoma.

BACKGROUND: Interleukin 8 (IL-8) is a pro-angiogenic, pro-inflammatory mediator that belongs to the family of chemokines. Due to its pro-angiogenic characteristic, it may play a vital role in tumour angiogenesis and progression. OBJECTIVES: This study was designed to estimate the levels of salivary IL-8 in oral precancer and oral squamous cell carcinoma (OSCC) patients and compare them with healthy controls. The aim was to evaluate its efficacy as a potential biomarker for these diseases. MATERIALS AND METHODS: Each group comprised 25 individuals. The salivary IL-8 levels were determined by enzyme-linked immunosorbent assay. RESULTS: The levels of salivary IL-8 were found to be significantly elevated in patients with OSCC as compared to the precancer group (p < 0.0001) and healthy controls (p < 0.0001). However, the difference in salivary IL-8 concentrations among the precancer group and controls was statistically non-significant (p = 0.738). CONCLUSIONS: Our results suggested that salivary IL-8 can be utilised as a potential biomarker for OSCC. Salivary IL-8 was found to be non-conclusive for oral premalignancy in this preliminary study. Hence, its possible role in transition from premalignancy to malignancy needs further research with larger sample sizes. CLINICAL RELEVANCE: Saliva as a diagnostic biofluid offers a number of advantages over blood-based testing. The role of IL-8 in oral cancer if validated further by future research can provide an easy diagnostic test as well as a prognostic indicator for patients undergoing treatment. Therefore, if it's role in tumourigenesis can be sufficiently assessed, it could open up new avenues to find out novel treatment modalities for oral cancer.

Langerhans cells and T cells sense cell dysplasia in oral leukoplakias and oral squamous cell carcinomas--evidence for immunosurveillance.

Leukoplakias (LPLs) are lesions in the oral mucosa that may develop into oral squamous cell carcinoma (OSCC). The objective of this study was to assess presence and distribution of dendritic Langerhans cells (LCs) and T cells in patients with LPLs with or without cell dysplasia and in oral squamous cell carcinoma (OSCC). Biopsy specimens from patients with leukoplakias (LPLs) with or without dysplasia and oral squamous cell carcinoma (OSCC) were immunostained with antibodies against CD1a, Langerin, CD3, CD4, CD8 and Ki67, followed by quantitative analysis. Analyses of epithelium and connective tissue revealed a significantly higher number of CD1a + LCs in LPLs with dysplasia compared with LPLs without dysplasia. Presence of Langerin + LCs in epithelium did not differ significantly between LPLs either with or without dysplasia and OSCC. T cells were found in significantly increased numbers in LPLs with dysplasia and OSCC. The number of CD4+ cells did not differ significantly between LPLs with and without dysplasia, but a significant increase was detected when comparing LPLs with dysplasia with OSCC. CD8+ cells were significantly more abundant in OSCC and LPLs with dysplasia compared with LPLs without dysplasia. Proliferating cells (Ki67+) were significantly more abundant in OSCC compared to LPLs with dysplasia. Confocal laser scanning microscopy revealed colocalization of LCs and T cells in LPLs with dysplasia and in OSCC. LCs and T cells are more numerous in tissue compartments with dysplastic epithelial cells and dramatically increase in OSCC. This indicates an ongoing immune response against cells with dysplasia.

[Study of testicular cancer gene expression in samples of oral leukoplakia and squamous cell carcinoma of the mouth].

Cancer-testis (CT) antigens are normally expressed mostly in human germ cells, there is also an aberrant expression in some tumor cells. This expression profile makes them potential tumor growth biomarkers and a promising target for tumor immunotherapy. Specificity of CT genes expression in oral malignant and potentially malignant diseases, e.g. oral leukoplakia, is not yet studied. Data on CT genes expression profile in leukoplakia would allow developing new diagnostic methods with potential value for immunotherapy and prophylaxis of leukoplakia malignization. In our study we compared CT genes expression in normal oral mucosa, oral leukoplakia and oral squamous cell carcinoma. We are the first to describe CT genes expression in oral leukoplakia without dysplasia. This findings make impossible differential diagnosis of oral leukoplakia and squamous cell carcinoma on the basis of CT genes expression. The prognostic value of CT genes expression is still unclear, therefore the longitudinal studies are necessary.

TWIST and p-Akt immunoexpression in normal oral epithelium, oral dysplasia and in oral squamous cell carcinoma.

OBJECTIVES: The aim of this study was to evaluate the immunoexpression of TWIST and p-Akt proteins in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC), correlating their expressions with the histological features of the lesions. STUDY DESIGN: Immunohistochemical studies were carried out on 10 normal oral epithelium, 30 OL and 20 OSCC formalin-fixed, paraffin-embedded tissue samples. Immunoperoxidase reactions for TWIST and p-Akt proteins were applied on the specimens and the positivity of the reactions was calculated for 1000 epithelial cells. RESULTS: Kruskal-Wallis and Dunn's post tests revealed a significant difference in TWIST and p- Akt immunoexpression among normal oral mucosa, OL and OSCC. In addition, a significant positive correlation was found between TWIST and p-Akt expressions according to the Pearson's correlation test. CONCLUSIONS: The results obtained in the current study suggest that TWIST and p-Akt may participate of the multi-step process of oral carcinogenesis since its early stages.