Nivolumab for Patients With High-Risk Oral Leukoplakia: A Nonrandomized Controlled Trial.

Importance: Proliferative verrucous leukoplakia (PVL) is an aggressive oral precancerous disease characterized by a high risk of transformation to invasive oral squamous cell carcinoma (OSCC), and no therapies have been shown to affect its natural history. A recent study of the PVL immune landscape revealed a cytotoxic T-cell-rich microenvironment, providing strong rationale to investigate immune checkpoint therapy. Objective: To determine the safety and clinical activity of anti-programmed cell death 1 protein (PD-1) therapy to treat high-risk PVL. Design, Setting, and Participants: This nonrandomized, open-label, phase 2 clinical trial was conducted from January 2019 to December 2021 at a single academic medical center; median (range) follow-up was 21.1 (5.4-43.6) months. Participants were a population-based sample of patients with PVL (multifocal, contiguous, or a single lesion ≥4 cm with any degree of dysplasia). Intervention: Patients underwent pretreatment biopsy (1-3 sites) and then received 4 doses of nivolumab (480 mg intravenously) every 28 days, followed by rebiopsy and intraoral photographs at each visit. Main Outcomes and Measures: The primary end point was the change in composite score (size and degree of dysplasia) from before to after treatment (major response [MR]: >80% decrease in score; partial response: 40%-80% decrease). Secondary analyses included immune-related adverse events, cancer-free survival (CFS), PD-1 ligand 1 (PD-L1) expression, 9p21.3 deletion, and other exploratory immunologic and genomic associations of response. Results: A total of 33 patients were enrolled (median [range] age, 63 [32-80] years; 18 [55%] were female), including 8 (24%) with previously resected early-stage OSCC. Twelve patients (36%) (95% CI, 20.4%-54.8%) had a response by composite score (3 MRs [9%]), 4 had progressive disease (>10% composite score increase, or cancer). Nine patients (27%) developed OSCC during the trial, with a 2-year CFS of 73% (95% CI, 53%-86%). Two patients (6%) discontinued because of toxic effects; 7 (21%) experienced grade 3 to 4 immune-related adverse events. PD-L1 combined positive scores were not associated with response or CFS. Of 20 whole-exome sequenced patients, all 6 patients who had progression to OSCC after nivolumab treatment exhibited 9p21.3 somatic copy-number loss on pretreatment biopsy, while only 4 of the 14 patients (29%) who did not develop OSCC had 9p21.3 loss. Conclusions and Relevance: This immune checkpoint therapy precancer nonrandomized clinical trial met its prespecified response end point, suggesting potential clinical activity for nivolumab in high-risk PVL. Findings identified immunogenomic associations to inform future trials in this precancerous disease with unmet medical need that has been difficult to study. Trial Registration: ClinicalTrials.gov Identifier: NCT03692325.

Nicotine promotes the development of oral leukoplakia via regulating peroxiredoxin 1 and its binding proteins.

Tobacco can induce reactive oxygen species (ROS) production extensively in cells, which is a major risk factor for oral leukoplakia (OLK) development. Peroxiredoxin 1 (Prx1) is a key antioxidant protein, upregulated in a variety of malignant tumors. We previously found that nicotine, the main ingredient of tobacco, promotes oral carcinogenesis via regulating Prx1. The aim of the present study was to screen and identify the Prx1 interacting proteins and investigate the mechanisms of nicotine on the development of OLK. Through liquid chromatography-tandem mass spectrometry combined with bioinformatics analysis, the candidate Prx1 interacting proteins of cofilin-1 (CFL1), tropomyosin alpha-3 chain (TPM3), and serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform (PPP2R1A) were screened in human dysplastic oral keratinocyte cells treated with nicotine. CFL1, TPM3, and PPP2R1A were highly expressed in human OLK tissues. The expression of CFL1 increased and the expression of PPP2R1A decreased in OLK of smokers compared to that in OLK of non-smokers. Nicotine upregulated CFL1 and downregulated PPP2R1A in 4-nitro-quinoline-1-oxide (4NQO)-induced OLK tissues in mice in part dependent on Prx1. Furthermore, the in-situ interaction of CFL1, TPM3, and PPP2R1A with Prx1 were validated in human OLK tissues. Our results suggested that tobacco might promote the development of OLK via regulating Prx1 and its interacting proteins CFL1 and PPP2R1A.

Nicotine promotes the development of oral leukoplakia via regulating peroxiredoxin 1 and its binding proteins

Tobacco can induce reactive oxygen species (ROS) production extensively in cells, which is a major risk factor for oral leukoplakia (OLK) development. Peroxiredoxin 1 (Prx1) is a key antioxidant protein, upregulated in a variety of malignant tumors. We previously found that nicotine, the main ingredient of tobacco, promotes oral carcinogenesis via regulating Prx1. The aim of the present study was to screen and identify the Prx1 interacting proteins and investigate the mechanisms of nicotine on the development of OLK. Through liquid chromatography-tandem mass spectrometry combined with bioinformatics analysis, the candidate Prx1 interacting proteins of cofilin-1 (CFL1), tropomyosin alpha-3 chain (TPM3), and serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform (PPP2R1A) were screened in human dysplastic oral keratinocyte cells treated with nicotine. CFL1, TPM3, and PPP2R1A were highly expressed in human OLK tissues. The expression of CFL1 increased and the expression of PPP2R1A decreased in OLK of smokers compared to that in OLK of non-smokers. Nicotine upregulated CFL1 and downregulated PPP2R1A in 4-nitro-quinoline-1-oxide (4NQO)-induced OLK tissues in mice in part dependent on Prx1. Furthermore, the in-situ interaction of CFL1, TPM3, and PPP2R1A with Prx1 were validated in human OLK tissues. Our results suggested that tobacco might promote the development of OLK via regulating Prx1 and its interacting proteins CFL1 and PPP2R1A.

Oral leukoplakia, a precancerous lesion of squamous cell carcinoma, in patients with long-term pegylated liposomal doxorubicin treatment.

Pegylated liposomal doxorubicin (PLD) has a good safety profile, but long-term use has been associated with development of squamous cell carcinoma of the tongue and oral cavity (SCCTO) in some patients. The study objective was to estimate the prevalence of oral leukoplakia, a known precursor of SCCTO, in patients with ovarian cancer and long-term PLD use.After approval of the institutional review board, medical record of 114 patients who were treated with PLD at our institution between January 2010 and December 2016 were retrospectively reviewed. All those patients have been referred for routine monitoring of oral mucositis every time before administration by a dentist. The patient characteristics included in the evaluation were age, smoking and drinking habits, the PLD dose and schedule, and presence or absence of oral leukoplakia and SCCTO at each oral examination. The relationships of the incidence of oral leukoplakia and patient characteristics were analyzed.The median total PLD dose was 160 (range 40-1550) mg/m. Oral leukoplakia was seen in 6 (5.3%) patients. The median PLD dose, at the time of oral leukoplakia diagnosis, was 685 (range 400-800) mg/m. SCCTO was not found. Univariate analysis revealed that age, Brinkman index, and habitual drinking were not considered as risk factors for oral leukoplakia, and only total PLD dose (OR, 1.470; 95% CI, 1.19-1.91; P < .001) remained as a significant independent risk factor for oral leukoplakia. The ROC curve analysis indicated that the optimal cutoff value of the total PLD dose to predict development of oral leukoplakia was 400 mg/m. The sensitivity was 100% and the specificity was 88.8%. No patient discontinued PLD because of oral leukoplakia or SCCTO.The 2 most important clinical observations were the occurrence of oral leukoplakia in patients with long-term PLD use and that the development of oral leukoplakia was related to a total cumulative dose ≥400 mg/m. Routine oral surveillance is recommended, particularly when the cumulative total dose exceeds 400 mg/m.

The influence of sleep restriction on expression of apoptosis regulatory proteins p53, Bcl-2 and Bax following rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide.

PURPOSE: The aim of this study was to evaluate whether sleep restriction (SR) could affect the mechanisms and pathways' essentials for cancer cells in tongue cancer induced by 4-nitroquinoline 1-oxide in Wistar rats. METHODS: The animals were distributed into 4 groups of 5 animals each treated with 50 ppm 4 NQO solution through their drinking water for 4 and 12 weeks. The animals were submitted to sleep restriction for 21 days using the modified multiple platform method, which consisted of placing 5 rats in a cage (41 × 34 × 16 cm) containing 10 circular platforms (3.5 cm in diameter) with water 1 cm below the upper surface. The investigations were conducted using immunohistochemistry of p53, Bax and Bcl-2 proteins related to apoptosis and its pathways. RESULTS: Although no histopathologic abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure in all groups, in 12 weeks were observed pre-neoplastic lesions. Data analysis revealed statistically significant differences (P < 0.05) in 4 weeks group for p53, and for bcl-2. Following 12 weeks of 4NQO administration, we found significant differences between SR and control groups in p53, bax, and bcl-2 immunoexpression. CONCLUSION: Our results reveal that sleep restriction exerted alterations in proteins associated with proliferation and apoptosis in carcinogenesis.

Systemic toxic effects during early phases of topical 4-NQO-induced oral carcinogenesis in rats.

BACKGROUND: Most studies have demonstrated 4-NQO toxicity to oral epithelium during oral carcinogenesis induction, but systemic toxicity has been poorly addressed. The aim of this study was to describe the systemic effect of 4-NQO topical application during early phases of oral cancer induction. METHODS: A 4-NQO propylene glycol ointment was topically applied on the rat tongue three times a week for 16 weeks. Local and systemic 4-NQO toxicity was evaluated by body weight gain, hematology, and serum chemistry analyses, histopathology, and proliferating cell nuclear antigen (PCNA) immunohistochemistry. RESULTS: Significant reduction in body weight gain and in white blood cell count as well as significant increase in serum ALT and AST was observed after 16 weeks of 4-NQO topical application. Focal hepatic lobular necrosis, renal tubular degeneration, and decreased cellularity in the splenic white pulp were also detected. CONCLUSIONS: 4-NQO topical application on the tongue of rats for 16 weeks seems to have caused hepatic, renal, and splenic toxicity. Potential systemic toxicity should be considered to monitor for variables that could interfere in topical oral carcinogenesis experiments.

Homeobox C5 expression is associated with the progression of 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis.

BACKGROUND: Aberrant expression of homeobox genes (HOX), normally required for the differentiation of a particular tissue, has been reported in several types of cancer, but poorly addressed in oral squamous cell carcinoma (OSCC). The present study investigated the expression of HOXC5 in OSCC and identified molecular biomarker whose expression is associated with the multistep oral carcinogenesis. METHODS: The expression of HOXC5, proliferation cell nuclear antigen (PCNA), and Bcl-2 was examined by RT-PCR and Western blot analysis and confirmed by immunohistochemistry and transferase-mediated dUTP nick end-labeling (TUNEL) assay in a 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis model. RESULTS: Homeobox genes C5 was overexpressed in SCC tissues, but not in normal tissues by RT-PCR and Western blot analysis. Along with the progress of multistep carcinogenesis, the levels of HOXC5 expression of mRNA and protein significantly increased during the dysplasia (moderate to severe dysplasia) when compared with normal and hyperplasia. The levels of PCNA and Bcl-2 were sequentially increased from hyperplasia to dysplasia and SCC. By immunohistochemistry, HOXC5 expression was significantly increased in dysplasia, whereas PCNA expression was gradually increased during tongue carcinogenesis. TUNEL-positive cells were increased until dysplasia, but reduced in SCC. CONCLUSIONS: These results indicate that overexpression of HOXC5 is correlated with oral carcinogenesis and strongly contributed to the development of OSCC. HOXC5 may be a useful biomarker and has an emerging therapeutic target of OSCC.

Prevalence of oral mucosal abnormalities in addiction treatment centre residents in Southern Ireland.

This study examines the prevalence of oral mucosal lesions and conditions among Irish addiction treatment centre residents and explores the feasibility and acceptability of a targeted oral cancer screening programme for such individuals. Four alcohol addiction treatment centres were visited periodically over a 12-month period. Two hundred and twenty residents (78% of 283 targeted) were interviewed regarding their alcohol, tobacco and drug habits (type, quantity, duration), and attitudes to dental care. Comprehensive oral examinations were performed. All potentially sinister soft tissue lesions/symptoms were referred for further investigation. Data analysis utilised SPSS-18. Ten participants who denied a history of alcohol/drug addiction were excluded from the main study. Remaining 210 participants comprised 148 males (70%) and 62 females (30%), ranging from 18 to 73 years of age, (mean 37.65; S.D. 13.82); 60% were under 40. High rates of tobacco and alcohol usage were recorded, 53% reported dual addiction (drug+alcohol), 44% alcohol only, 3% drug only. The prevalence of mucosal abnormalities was 29% with 84 mucosal abnormalities/symptoms detected in 61 subjects, comprising 28 extra-oral lesions/symptoms and 56 intra-oral lesions. Residents with mucosal abnormalities were significantly older (mean 41.8 years; S.D. 14.3) than those without such lesions (mean 35.95; S.D. 13.3), (p<0.05). Highest prevalences were noted for candidiasis (3.8%), facial scaring/laceration (3.8%), intra-oral lumps/swellings (2.9%), lymphadenopathy (2.9%) and hoarseness (1.9%). Four red areas suggestive of erythroplasia and two leukoplakic lesions were detected. Study addresses the paucity of data on the prevalence of oral mucosal lesions in addicted persons in Southern Ireland. Thirteen extra-oral lesions/symptoms and 19 intra-oral lesions were potentially significant. Despite the relatively poor follow-up compliance rate (33%), two premalignant lesions were confirmed in the main study group, yielding a detection rate of 0.9%. Results suggest that an oral cancer screening programme targeted at individuals in addiction treatment centres may provide a feasible way to access persons with a history of tobacco and alcohol abuse. A high rate of untreated disease and emergency only attendance was seen in this study suggesting a lack of engagement with GDP services. Opportunistic screening in primary care is therefore unlikely to capture this cohort. Inclusion of oral cancer screening in the routine medical examination given to residents of addiction treatment centres may provide an efficient and effective way to detect potentially malignant lesions in these high-risk individuals.

Betel-quid chewing with or without tobacco is a major risk factor for oral potentially malignant disorders in Sri Lanka: a case-control study.

We investigated the prevalence of, and risk factors for, oral potentially malignant disorders (OPMDs) in rural Sri Lanka. A cross-sectional community-based study was conducted by interview and oral examination of 1029 subjects aged over 30 years. A community-based nested case-control study then took those with OPMDs as 'cases', "controls" being those with no oral abnormalities at time of initial screening. The prevalence of OPMD was 11.3% (95% CI: 9.4-13.2), after weighting for place of residence and gender. Risk factors were betel-quid (BQ) chewing daily [OR=10.6 (95% CI: 3.6-31.0)] and alcohol drinking daily or weekly [OR=3.55 (1.6-8.0)]. A significant dose-response relationship existed for BQ chewing. Smoking did not emerge when adjusted for covariates. A synergistic effect of chewing and alcohol consumption existed. The attributable risk (AR) of daily BQ chewing was 90.6%, the population AR 84%. This study demonstrates high prevalence of OPMD, betel-quid chewing with or without tobacco being the major risk factor.

Cyclin D1 expression and its possible regulation in chewing tobacco mediated oral squamous cell carcinoma progression.

OBJECTIVE: Proto-oncogene cyclin D1 is a G1 phase specific cell cycle regulator and known for its role in various cancers. The aim of the study was to understand oral cancer progression by observing the mRNA and protein expression of cyclin D1. METHODS: Different oral tissue samples were selected as a model to study oral cancer progression. Those include healthy oral mucosa, premalignant lesions (Leukoplakia, Erythroplakia, Oral SubMucous Fibrosis) and oral cancer (OSCC) samples. Cyclin D1 mRNA and protein expression were detected by slot-blot and by immunohistochemical methods, respectively. RESULTS: Premalignant lesions (PML) showed average 3-fold increase in the mRNA expression than normal oral mucosa (p = 0.001) whereas only 1.3-fold increase in mRNA has been observed in OSCC samples over the PML. On the other hand OSCC showed average 4-fold increase in mRNA expression than normal oral mucosa (p < 0.001). Cyclin D1 protein accumulation has been observed in 31.3% (16/51) of the OSCC samples whereas the normal oral mucosa and the PML showed no immunoreactivity. Oral cancer samples showing positive cyclin D1 immunoreactivity has increased from 15.0% (3/20) well differentiated SCC to 31.2% (5/16) moderately differentiated SCC to 53.3% (8/15) poorly differentiated SCC, found statistically significant (p = 0.05). CONCLUSION: By observing the expression of cyclin D1 in different stages, we have noticed two major transitions that occur in normal oral mucosa that leads to oral cancer. The first transitional event transforms the normal oral mucosa to PML whereas the second transition drives the PML to OSCC. These findings give evidence that the first transition induces cyclin D1 mRNA with no detectable cyclin D1 protein. The induction of mRNA is maintained with increased cyclin D1 protein accumulation in the second transition.

Increased expression of focal adhesion kinase correlates with cellular proliferation and apoptosis during 4-nitroquinoline-1-oxide-induced rat tongue carcinogenesis.

BACKGROUND: Oral carcinogenesis is a multistep process and requires accumulation and interplay of a series of molecular genetic events. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in signalling pathways that are initiated at sites of integrin-mediated cell adhesions and by growth factor receptors. Overexpression of FAK has been linked to oral squamous cell carcinoma (OSCC). So, it is hypothesized that FAK expression might contribute to oral carcinogenesis. METHODS: During 4-nitroquinoline-1-oxide-induced rat tongue carcinogenesis, FAK protein expression, proliferating cell nuclear antigen (PCNA) and apoptotic nuclei (TUNEL assay) were examined by means of immunohistochemistry. RESULTS: Along with the progress of multistage carcinogenesis, FAK expression increased significantly among different histopathological groups with normal mucosa, mild-dysplastic epithelia, moderate-dysplastic epithelia, severe-dysplastic epithelia and in turn OSCC. Furthermore, FAK immunohistochemical index and PCNA-labelling index displayed positive correlation (r = 0.946, P < 0.05), while negative associations were revealed between apoptotic index and final FAK index (r = -0.959, P < 0.05). CONCLUSION: Our results implicated a role for FAK in oral carcinogenesis. Inhibition of FAK might be a potential novel treatment strategy in this disease.

The effect of betel quid and cigarette on multistate progression of oral pre-malignancy.

BACKGROUND: Although the effect of betel quid chewing and smoking on oral cancer has been well documented, both influences, dose and duration during life time, on multistate progression of oral pre-malignancy are hardly addressed. METHODS: By recruiting a group of male chewers from different occupation groups, we modeled the effects of both duration and quantity of betel quid chewing and smoking on annual incidence rate of developing leukoplakia and average dwelling times (ADTs) staying in leukoplakia and erythroleukoplakia. RESULTS: The annual incidence rate (per year) of leukoplakia was estimated as 0.35% (95% CI: 0.22-0.48%). The ADTs were 24 years for leukoplakia and 7 years for erythroleukoplakia. Annual incidence rate of leukoplakia with high consumption and long duration of betel quid and smoking was higher. Both quantity and duration of smoking and betel quid chewing play minor roles in the influence of ADT. The risks of developing oral cancer after 20 years of follow-up were 42.2% for leukoplakia and 95.0% for erythroleukoplakia. CONCLUSION: The effects of betel quid chewing and smoking on multistate progressions between oral pre-malignancies were elucidated. These results can be applied to predict long-term risk of malignant transformation varying with different duration and quantity of betel quid and cigarette.

A review of betel quid chewing, oral cancer and precancer in Mainland China.

On the Chinese mainland, betel quid (BQ) chewing is common in the Hunan and Hainan provinces. The BQ chewing habit in Hunan consists of dried husks and betel nuts, which are sold as industrially packaged, areca nut-based products. In Hainan, the fresh nut is chewed. Tobacco is not added. Reported prevalence of BQ chewing in Hunan province is high (64.5-82.7%). Oral diseases associated with BQ chewing are oral submucous fibrosis (OSF), oral leukoplakia (OL) and oral cancer. Reported prevalence of OSF among BQ chewers ranges from 0.9% to 4.7%. People most commonly affected are between the ages of 30 and 39 years, and 40 and 49 years. The reported prevalence of OL in Hainan ranges from 2.1% to 2.5%. In BQ chewers who also smoke, the reported prevalence is 20.3%. The prevalence of OL in Hunan province ranges from 0.1% to 0.5%. The prevalence of oral cancer among BQ chewers is low, ranging from 0.02% to 0.05%. In cases of OSF, reported prevalence is 2.6% and 1.2%. Presently, data on prevalence of BQ chewing in southern provinces of Mainland China is limited. BQ chewing habits, however, seem to differ between geographic areas. Future case-control studies are necessary to evaluate the risk for oral cancer and other associated oral mucosal diseases resulting from variations in BQ chewing habits.

Clinical, pathological, cellular and molecular lesions caused by oral smokeless tobacco--a review.

While carcinogenicity of smokeless tobacco (ST) to humans is well established the oral lesions that precede development of cancer are less well characterized. The clinical appearances of ST-associated lesions are variable. Epidemiological studies show a strong significant association of risk with chronic daily use but population differences are noted because of various commercial products in use. Morphological features observed are some what different to oral lesions caused by smoking and oral dysplasia in ST-associated lesions is less common. Effects of ST on oral keratinocytes observed in vitro include alterations in cell proliferation, apoptosis and activation of inflammatory markers. Genetic aberrations caused by ST include activation of ras, uncommon in smokers but mutational hot spots in p53 encountered are similar to those in smokers.

Expression of ets-1 is not affected by N-ras or H-ras during oral oncogenesis.

PURPOSE: To determine whether ras-activated cascades lead to activation of ets-1 expression in sequential histological stages of oral oncogenesis in an experimental animal model. METHODS: Thirty-seven Syrian golden hamsters were divided into three experimental groups (A, B, C) and one control group. The hamsters' buccal pouches in experimental groups were treated with 0.5% 9, 10-dimethyl-1, 2-benzanthracene (DMBA) for 14 weeks and were excised at 10, 14, 19 weeks, respectively. The biopsies were classified pathologically (normal mucosa, hyperkeratosis, hyperplasia, dysplasia, early invasion, well and moderately differentiated carcinoma) and studied immunohistochemically. The two-tailed Student's t test was performed for each animal group and for each histological category. RESULTS: The ets-1 expression increased in early stages of oral tumor formation and invasion. The expression of N-ras gradually decreased during oral oncogenesis, as previously observed with H-ras. CONCLUSIONS: Neither N-ras nor H-ras affects ets-1 expression in contrast to other types of cancer in which N-ras and ets-1 are implicated in the same signalling pathway. Therefore, the existing pathway implicating these proteins might be somehow altered in oral cancer. It seems that ets-1 is a good prognostic marker for invasiveness and progression of oral cancer.

Khat (Catha edulis)-an updated review.

The habit of chewing fresh leaves and twigs of khat (Catha edulis) for their stimulating amphetamine-like effects is highly prevalent in East Africa and southwest on the Arabic peninsula. There is an extensive literature on khat providing information about its history, botany, production, geographical distribution, chemistry and pharmacology, and exploring the social, economic, medical, psychological and oral aspects related to its use. Some of this literature dates as early as the 11th century; however, most of it appeared after the first scientific description of khat by Peter Forskal in 1775. This review provides a panorama of khat and the various aspects of its use. A non-technical description of the plant chemistry and pharmacology is included. The medical, psychological and oral aspects are emphasized, and the current knowledge about the microbiological effects of khat is also presented.

Smokeless tobacco (khaini) extracts modulate gene expression in epithelial cell culture from an oral hyperplasia.

Smokeless tobacco (ST) usage is a growing public health problem worldwide. Exposure to smokeless tobacco is carcinogenic to humans. The molecular mechanism(s) underlying ST associated oral carcinogenesis remain largely unknown. The major challenge is to identify the key factor(s) involved in malignant transformation of oral lesions. Knowledge of these factors will provide candidate diagnostic biomarkers and targets for early intervention. To identify the molecular targets in ST associated oral lesions, we established and purified cultures of epithelial cells (AMOL-III) from an oral leukoplakia with histological evidence of hyperplasia with hyperkeratosis from gingivo-buccal sulcus of a smokeless tobacco (khaini) consumer. Cell cultures were characterized and modulation of gene expression in response to smokeless tobacco extract (STE) was investigated using confocal microscopy and immunoblotting. AMOL-III cells showed altered expression of cell cycle regulators namely p53, p21waf1/cip1, hdm2, proliferation marker Ki67 and transcription factor Ets-1. These cells did not harbor HPV 16/18. No mutation was detected in H-Ras codon 12/13 or in p53 exons 5-9 in AMOL-III cells. STE treatment of these cells resulted in loss of pRb, RARbeta, p21 waf1/cip1 and O6-methyl guanine-DNA methyl transferase (MGMT) while the expression of cyclin D1 was increased. To our knowledge this is the first report to demonstrate that khaini modulates expression of multiple cellular targets including proteins involved in cell cycle regulation and DNA methylation, which may lead the oral epithelial cells down the carcinogenic pathway. This in vitro model system assumes importance in unraveling the cellular and molecular mechanisms implicated in smokeless tobacco associated early oral cancer progression.

Immunohistochemical assessment of Viadent-associated leukoplakia.

Studies show an association between sanguinarine, the active ingredient in Viadent oral health care products, and oral premalignant lesions. The study was undertaken to quantitatively compare the staining profiles of sanguinarine-associated leukoplakia to normal and dysplastic specimens. Archived oral mucosal specimens were stained for tumor markers p16, p53, cyclin D1, Ki-67, and Bcl-x and analyzed through Simple PCI image analysis software. Quantitative analyses showed trends towards intermediate staining in Viadent-related specimens (Ki-67: normal: 18.12+/-2.15, Viadent: 16.12+/-2.16, dysplasia: 14.53+/-2.04, p>0.05; cyclin D1: normal: 15.65+/-3.68, Viadent: 12.52+/-3.57, dysplasia: 1.94+/-3.93, p<0.05; p16: normal: 55.04+/-4.16, Viadent: 49.74+/-4.16, dysplasia: 45.03+/-4.45; p>0.05; p53:normal: 2.65+/-1.37, Viadent: 4.64+/-1.52, dysplasia: 8.71+/-1.37; p<0.05 Kruskal Wallace, Tukey/Kramer). Our Viadent profiles, intermediate between normal and dysplasia, support a preneoplastic nature of this process.