Expression patterns of cancer stem cell markers ALDH1 and CD133 correlate with a high risk of malignant transformation of oral leukoplakia.

Molecular markers for predicting oral cancer development in premalignant oral leukoplakia (OL) are urgently needed. The objective of this study was to examine the expression patterns of cancer stem cell markers ALDH1 and CD133 in samples from patients with OL, and determine their prognostic values for subsequent development of oral cancer. Immunohistochemistry for ALDH1 and CD133 was performed in samples from a cohort of 141 patients with biopsy-proven OL who received a mean follow-up of 5.5 years. Patient clinicopathologic and follow-up data were analyzed. Expression of ALDH1 and CD133 was observed in 54 (38.3%) and 32 (22.7%) of 141 patients with OL, respectively. Kaplan-Meier analysis showed that 48.1% patients with ALDH1-positivity developed oral cancer compared with 12.6% those with ALDH1-negativity (p < 0.001). Meanwhile, 59.4% patients with CD133-positivity developed oral cancer compared with 16.5% those with CD133-negativity (p < 0.001). Multivariate analysis revealed that ALDH1 and CD133 expression was associated with 4.17-fold [95% confidence interval (CI), 1.96-8.90; p < 0.001] and 2.86-fold (95% CI, 1.48-5.55; p = 0.002) increased risk of OL transformation, respectively. Collectively, these data demonstrated for the first time that the expression of ALDH1 and CD133 correlated with malignant transformation in a large series of patients with OL who received a long-term follow-up, which suggests that they may serve as predictors to identify OL with a high risk of oral cancer development.

Phospholipase C-γ1 expression correlated with cancer progression of potentially malignant oral lesions.

BACKGROUND: Phospholipase C-γ1 (PLCγ1) is required for cellular migration during tumor progression and invasion of oral squamous cell carcinoma (OSCC) cells. The objective of the current study was to determine immunoexpression pattern of PLCγ1 in oral potentially malignant lesions (OPLs) and evaluate PLCγ1 usefulness as a biomarker for predicting clinical behavior in the carcinogenesis of OPL. METHODS: In a retrospective follow-up study, the expression pattern of PLCγ1 protein was determined using immunohistochemistry in samples from 68 patients, including untransformed cases (n = 38) and malignant-transformed cases (n = 30). The corresponding post-malignant lesions (OSCCs) were also performed. RESULTS: We observed that elevated expression of PLCγ1 in 40 of 68 (59%) general OPLs and 23 of 30 (77%) OSCCs compared with that in normal oral mucosa. Kaplan-Meier analysis revealed that patients with PLCγ1 positivity had a significantly higher incidence of OSCC than those with PLCγ1 negativity. Cox regression analysis revealed that PLCγ1 expression patterns were significantly associated with increased risk of malignant progression. In addition, the correlation between PLCγ1 expression in pre-malignant OPL and that in post-malignant OSCC was significant (P = 0.004). CONCLUSION: These data indicate that PLCγ1 expression in OPL correlated with oral cancer progression, and PLCγ1 may serve as a useful marker for the identification of high-risk OPL into OSCC.

Expression of cancer stem cell markers ALDH1 and Bmi1 in oral erythroplakia and the risk of oral cancer.

BACKGROUND: Oral erythroplakia (OE) is a notoriously aggressive oral pre-malignant lesion with a high tendency to oral cancer development, but its biological behavior is largely unknown. The objective of this study was to determine the expression of cancer stem cell markers ALDH1 and Bmi1 in OE and their correlation with malignant transformation of OE. METHODS: In a retrospective case-control study, expression patterns of ALDH1 and Bmi1 were determined using immunohistochemistry in samples from 34 patients with OE, including patients with untransformed lesions (n=17) and patients with malignant transformed lesions (n=17). RESULTS: ALDH1 and Bmi1 expression was observed in 19 (55.9%) and 20 (58.8%) of 34 patients with OE, respectively. Multivariate analysis revealed that ALDH1 expression was significantly associated with increased risk of transformation (P<0.05), but Bmi1 expression was not a significant marker (P > 0.05). Notably, the coexpression of both ALDH1 and Bmi1 was a strong indicator associated with 8.56-fold (95% confidence interval [CI], 1.74-42.17; P<0.01) for malignant transformation. Point prevalence analysis revealed that 78.6% (95% CI, 54.0-100) of the patient with coexpression of both ALDH1 and Bmi1 developed oral cancer. CONCLUSION: Our data indicated that the expression patterns of ALDH1 and Bmi1 in OE were associated with malignant transformation, suggesting that they may be valuable predictors for evaluating the risk of oral cancer.

Two stem cell markers, ATP-binding cassette, G2 subfamily (ABCG2) and BMI-1, predict the transformation of oral leukoplakia to cancer: a long-term follow-up study.

BACKGROUND: Although oral leukoplakia (OL) is the best-known potentially malignant disorder, the risk of OL malignant transformation is difficult to assess. ATP-binding cassette, G2 subfamily (ABCG2) and BMI-1 are stem cell markers that have been found to be associated with head and neck tumorigenesis. The objective of the current study was to evaluate the usefulness of ABCG2 and BMI-1 in predicting OL transformation. METHODS: In a retrospective cohort of 135 patients with OL from the study institution who had a mean follow-up of 5.5 years, 32 developed cancer between 1985 and 2008. The expression of ABCG2 and BMI-1 was determined using immunohistochemistry in samples from these patients, and included untransformed OL (n = 103) and malignant-transformed OL (n = 32). The association between protein expression and clinicopathological parameters and transformation was analyzed. RESULTS: Expression of ABCG2 and BMI-1 was observed in 58 (43.0%) and 44 (32.6%) of 135 patients, respectively. The correlation between ABCG2 and BMI-1 expression was significant (P = .024). Kaplan-Meier analysis revealed that 37.9% of patients with ABCG2 positivity developed cancer compared with 13.0% of patients with ABCG2 negativity (P = .014, log-rank test). Approximately 40.9% of patients with BMI-1 positivity developed cancer compared with 15.4% of patients with BMI-1 negativity (P = .029, log-rank test). Multivariate analysis revealed that ABCG2 and BMI-1 expression was associated with a 3.24-fold (95% confidence interval [95% CI], 1.31-7.98; P = .011) and 4.03-fold (95% CI, 1.59-10.26; P = .003) increased the risk of transformation, respectively. CONCLUSIONS: ABCG2 and BMI-1 expression was found to be associated with the development of oral cancer in a large cohort of patients with OL for whom long-term follow-up was available, which suggests that ABCG2 and BMI-1 may be used as predictors of OL transformation.

A clinicopathological study on verrucous hyperplasia and verrucous carcinoma of the oral mucosa.

BACKGROUND: Oral verrucous hyperplasia (VH) and verrucous carcinoma (VC) are two clinicopathologically distinctive oral verrucous lesions. The objective of this study was to investigate the clinicopathological features of the two verrucous lesions and estimate their relationship from China. METHODS: Retrospective review of two series of patients with histologically confirmed VH (n = 121) and VC (n = 56) between 1996 and 2009 in our hospital were conducted. RESULTS: The average age of VH was 58.5 years (ratio male:female = 1.37) with the tongue being the predominant site. The average age of VC was 64.3 years (ratio male:female = 1.15) with the lower lip being the predominant site. Multivariate analysis revealed that the elderly patient with verrucous lesion (≥60 years) was associated with 3.06-fold (P = 0.007) increased carcinoma risk compared with the non-elderly patient. The lesion located on lower lip was associated with 13.54-fold (P < 0.001) increased carcinoma risk compared with other sites. CONCLUSION: Clinicopathological features of VH and VC in China were elucidated. Elderly patient with oral verrucous lesion located on the lower lip correlates with higher risk of carcinoma.

A retrospective clinicopathological study on oral lichen planus and malignant transformation: analysis of 518 cases.

OBJECTIVE: To investigate the epidemiological and clinical characteristics of a relatively large cohort of patients with oral lichen planus (OLP) from eastern China. STUDY DESIGN: A total of 518 patients with histologically confirmed OLP in a long-term follow-up period (6 months-21.5 years) were retrospectively reviewed in our clinic. RESULTS: Of the 518 patients, 353 females and 165 males were identified. The average age at diagnosis was 46.3 years (range 9-81 years) with the buccal mucosa being the most common site (87.8%). At initial presentation, white lichen and red lichen was seen in 52.3% and 47.7% patients, respectively. Of these, 5 (0.96%) patients previously diagnosed clinically and histopathologically as OLP developed oral cancer. All of them were the females with no a history of smoking or alcohol use. CONCLUSIONS: Clinical features of eastern Chinese OLP patients were elucidated. Notably, approximately 1% of OLP developed into cancer, which provides further evidence of potentially malignant nature of OLP.

Malignant transformation of oral epithelial dysplasia: clinicopathological risk factors and outcome analysis in a retrospective cohort of 138 cases.

AIMS: To explore the usefulness of a new binary system of grading dysplasia proposed by the World Health Organization and to identify significant risk factors for malignant transformation in a long-term follow-up cohort of patients with oral epithelial dysplasia. METHODS AND RESULTS: A total of 138 patients with histologically confirmed oral dysplasia between 1978 and 2008 were reviewed retrospectively in our department. The mean follow-up period was 5.1 years. Of these dysplasias, 37 (26.8%) developed into cancer, with a mean duration of 4.6 years. Cox regression analysis revealed that high-grade dysplasia was an independent risk factor for transition, but age, gender, lesion site, diet habit, smoking and alcohol intake were not risk factors. High-grade dysplasia was associated with a 2.78-fold (95% confidence interval 1.44-5.38; P = 0.002) increased risk of transition, as compared with low-grade dysplasia. Consistently, high-grade dysplasia had a significantly higher incidence of malignancy than low-grade dysplasia by Kaplan-Meier analysis (log-rank test, P = 0.001). CONCLUSIONS: The utilization of high-grade dysplasia as a significant indicator for evaluating malignant transformation risk in patients with potentially malignant lesions is suggested; this may be helpful to guide treatment selection in clinical practice.

Malignant potential of oral and labial chronic discoid lupus erythematosus: a clinicopathological study of 87 cases.

AIMS: To investigate clinicopathological features and identify clinicopathological risk factors for the malignant transformation of oral and labial chronic discoid lupus erythematosus (DLE) in a relatively large number of patients from China. METHODS AND RESULTS: A total of 87 patients with clinical and histopathological diagnosis of DLE between 1993 and 2009 were reviewed retrospectively in our hospital. The average age at diagnosis was 51.7 years, with a male:female ratio of 1:1.8. The lower lip was the most common site (71.3%). We documented six DLE patients with malignant transformation. On univariate analysis, patients with high-risk dysplasia (P = 0.002) or aged >60 (P = 0.045) were associated with DLE malignant transformation, but gender, lesion site, smoking and alcohol intake were not risk factors. On multivariate analysis, high-risk dysplasia was a significant indicator for DLE malignant transformation. High-risk dysplasia was associated with a 14.24-fold [95% confidence interval (95% CI), 1.97-102.88; P = 0.008] increased risk of malignant transformation, compared with non/low-risk dysplasia. CONCLUSIONS: The utilization of high-risk dysplasia as a significant indicator for evaluating malignant transformation risk in patients with DLE is suggested, which may be helpful to guide treatment selection.

Malignant transformation of oral verrucous leukoplakia: a clinicopathologic study of 53 cases.

BACKGROUND: Oral verrucous leukoplakia (VL) is one of the non-homogenous oral leukoplakias. The objective of this study was to investigate the clinicopathologic features of VL and identify the clinicopathologic risk factors that might be associated with VL malignant transformation from China. METHODS: Among 1541 patients with oral leukoplakia, a total of 53 patients with clinical and histopathologic diagnosis of VL between 1996 and 2009 were reviewed retrospectively in our hospital. RESULTS: Of the 53 patients, 11 (20.8%) with VL were observed to develop cancer in the study period. The average age at diagnosis was 59.8 years with a male/female ratio of 1.7:1. Tongue was the predominant site (41.5%). Multivariate regression analysis revealed that the elderly patients (>65 years old) were associated with 8.36-fold [95% confidence interval (95% CI), 1.45-48.09; P = 0.017] increased risk of malignant transformation compared with the non-elderly patients. The lesion located on gingiva was associated with 20.81-fold (95% CI, 1.94-222.80; P = 0.012) increased risk of malignant transformation compared with tongue. However, the gender, smoking, alcohol intake, and epithelial dysplasia were not risk factors. CONCLUSION: Clinicopathologic features of VL in China were elucidated. The utilization of age and lesion site at diagnosis as significant factors for evaluating malignant transformation risk in patients with VL was suggested. Further studies are required to investigate the roles of the potential risk factors in the VL malignant transformation.

Malignant transformation of oral leukoplakia: a retrospective cohort study of 218 Chinese patients.

BACKGROUND: Oral leukoplakia (OL) is the best-known potentially malignant disorder. A new binary system to grade dysplasia was proposed by WHO, but the biological significance in predicting malignant transformation risk is unknown. The objective of this study is to estimate the rate of malignant transformation in a long-term follow-up cohort, explore the usefulness of the new binary system of grading dysplasia and identify significant risk factors of OL malignant transformation in China. METHODS: A total of 218 patients with clinical and histopathologic diagnosis of OL were retrospectively reviewed. They were selected among all archived files at the Department of Oral Mucosal Diseases, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. The mean follow-up period was 5.3 years. RESULTS: Among 218 cases, 39 (17.9%) OL patients developed oral cancer, with a mean duration of 5.2 years. Cox regression analysis revealed that dysplasia was an independent risk factor for OL malignant transformation, but age, gender, lesion site, diet habit, smoking and ethanol intake were not risk factors. High-risk dysplastic OL was associated with a 4.57-fold (95% confidence interval, 2.36-8.84; P < 0.001) increased risk of malignant transformation, compared with low-risk dysplasia. Consistent with this result, high-risk dysplastic OL had significantly higher malignant incidence than low-risk dysplasia, particularly during the first 2-3 years of follow-up, by Kaplan-Meier analysis (Log-rank test, P < 0.001). CONCLUSIONS: The new binary system's function in predicting OL malignant transformation risk was investigated in this survey. The utilization of high-risk dysplasia as a significant indicator for evaluating malignant transformation risk in patients with OL was suggested, which may be helpful to guide treatment selection in clinical practice.

Human papillomavirus genotypes and p16 expression in oral leukoplakia and squamous cell carcinoma.

Several studies have shown a broad variation in the prevalence of human papillomavirus (HPV) in oral leukoplakia (OLK) and oral squamous cell carcinoma (OSCC), whereas the relationship is less well-defined and specific HPV genotypes lack examination in OLK. In the present study, the role of HPV and surrogate p16 expression was investigated to explore the correlation and pathogenesis in OLK and OSCC. Polymerase chain reaction (PCR) and flow-through hybridization technology were utilized to detect HPV genotypes in oral exfoliated cells from 30 healthy volunteers, 103 OLK and 30 OSCC patients. Expression of p16 was assessed by immunohistochemistry (IHC) in biopsies from these OLK and OSCC, in addition to 15 normal oral mucosal tissues as the control group. The healthy controls showed 3.3% (1/30) HPV presence; In OLK and OSCC, the detection rate was 4.9% (5/103), 3.3% (1/30), respectively. No significant relationship between HPV and OLK or OSCC was observed when compared with the control group (P>0.05). All 6 HPV-positive OLK and OSCC cases had p16 overexpression. But the sensitivity of p16 IHC was poor, because 88.4% (38/43) of p16 over-expressed OLK were HPV negative. There was no statistical significance between HPV and the sex, age, site, alcohol consumption, or smoking. These findings suggested HPV had a low prevalence in OLK and OSCC. This suggests the detection of HPV genotypes by PCR in exfoliated cells combined with p16 IHC may be more accurate to represent HPV infection.

A metabonomic approach to the diagnosis of oral squamous cell carcinoma, oral lichen planus and oral leukoplakia.

Early diagnosis of oral squamous cell carcinoma (OSCC) and precursor lesions is an attractive strategy to decrease patient morbidity and mortality, but presently there are no satisfied diagnostic approaches. This study proposed a metabonomics-based diagnostic approach for OSCC and its precancerous lesions, including oral lichen planus (OLP) and oral leukoplakia (OLK). Saliva samples were collected from patients and healthy donors, and HPLC/MS analysis was performed to acquire metabolic profiles. Diagnostic model was then constructed with hierarchical principal component analysis (HPCA) and discriminate analysis algorithms. The results indicate that metabolic profiling can properly describe the pathologic characteristics of OSCC, OLP and OLK. HPCA combined with kernel fisher discriminant analysis achieved 100% accuracy in diagnosis of test samples, which is superior to direct principal component analysis and other modeling algorithms. The metabonomic approach based on the integral investigation of oral metabolites enables the detection of OSCC and precancerous lesions on noninvasive saliva samples. The proposed approach is noninvasive, efficient and low-cost, and it can be developed as a promising method for population-based screening of cancers and precancers in the oral cavity.

Osteopontin expression in oral lichen planus.

To explore circulation levels of osteopontin (OPN), tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1 from patients with oral lichen planus (OLP) for clinical application. A group of 26 subjects with OLP were compared with 26 sex- and age-matched control (NC) subjects. Local lesion tissue was examined for OPN by immunohistochemical analysis. And, serum OPN, proinflammatory TNF-alpha and TGF-beta1 levels were measured by enzyme-linked immunoabsorbent assay. The serum concentrations of OPN and TNF-alpha were significantly higher in OLP patients than the NC group (P < 0.05). Although serum concentrations of TGF-beta1 increased slightly, they were not statistically significant. Erosive-form OLP exhibited significantly elevated TGF-beta1 serum levels, compared with reticular-form OLP. The above results suggest that the production of OPN is associated with the inflammatory process of OLP development, and may serve as a potential disease marker of OLP.

Alterations of GPI transamidase subunits in head and neck squamous carcinoma.

BACKGROUND: GPI anchor attachment is catalyzed by the GPI transamidase (GPIT) complex. GAA1, PIG-T and PIG-U are the three of five GPIT subunits. Previous studies demonstrated amplification and overexpression of GPIT subunits in bladder and breast cancer with oncogenic function. We performed an analysis of these subunits in head and neck squamous cell carcinoma (HNSCC). RESULTS: To evaluate GAA1, PIG-T and PIG-U in HNSCC, we used quantitative PCR (QPCR) and quantitative RT-PCR (QRT-PCR) to determine the copy number of those genes in primary tumors and the matching lymphocytes in 28 patients with HNSCC and quantified RNA expression of those genes in 16 primary HNSCC patients and 4 normal control tissue samples. GAA1 showed a significant increase in normalized mRNA expression, 2.11 (95% CI: 1.43, 2.79), in comparison to that of normal controls, 0.43 (95% CI: -0.76, 1.61), p = 0.014 (Mann-Whitney test). The mean genomic copy number of GAA1 was significantly increased in HNSCC, 0.59 (95% CI: 0.50, 0.79), in comparison to lymphocyte DNA, 0.35 (95% CI: 0.30, 0.50), p = 0.001 (paired t-test). CONCLUSION: An increased expression level and elevated copy number for GAA1 suggest a role for this GPI anchor subunit in HNSCC.

[Plasmid elimination effect of usnic acid on antibiotic-resistant Staphylococcus aureus].

OBJECTIVE: To observe the influence of Usnic acid on the antibiotic-resistant plasmid in Staphylococcus aureus (Sa). METHODS: The antibiotic-resistant plasmid was abstracted from the clinical divided strain of Sa and plasmid elimination test was performed in vitro. RESULTS: Plasmid elimination test showed that Usnic acid could eliminate the resistant plasmid in Sa effectively. At 24th and 48th hour after the treatment of Usnic acid, the elimination rate of resistant plasmid was 5.2% and 16.4% respectively. CONCLUSION: Usnic acid can eliminate the antibiotic-resistant plasmid in Sa. It is possible to use Usnic acid to treat the infection of antibiotic-resistant Sa in clinic.

[The effect of icariin and astragalosid I on the proliferation and differentiation of bone marrow stromal cells].

OBJECTIVE: To observe the effects of icariin and astragalosid I on the proliferative and alkaline phosphatase (ALP) activity of dog bone marrow stromal cells (BMSCs). METHODS: The dog's BMSCs were isolated and cultured in vitro. The 3th generation BMSCs were treated with icariin or astragalosid I at the concentration of 50 ng/ml and compared with BMSCs of BMP-2 group and control group. The growth curves of BMSCs were drawn by 3-(4,5-dimiethylthiazole-2-yl)-2, 5-hiphenyl tetrazolium bromide (MTT) colorimetric assay every day from the 1st to the 8th day to estimate the proliferative ability of BMSCs. The curves of OD value of ALP excreted by BMSCs on the 1st, 3th, 6th, 10th and the 14th day were recorded to estimate the ALP activity of BMSCs. RESULTS: After the pertreatment with icariin and astragalosid I, the BMSCs acquired higher MTF values and higher ALP's OD values as compared with control group and the difference between experiment group and control group was statistically significant (P < 0.05). CONCLUSION: Icariin and Astragealosid I can accelerate the proliferation and ALP excretion of BMSCs. At the same time, the osteogenesis ability of these cells is greatly improved.

[Research progress of quantitative DNA analysis in oral carcinoma and precancerous lesions].

Normal individual cells had 23 pairs of chromosome and stable DNA content. DNA content was varied during malignant transformation, which was specific feature of tumor. Quantitative DNA analysis can reflect cellular physiological or pathological condition by nuclear DNA content, which had significant role in early diagnosis, predication of prognosis and treatment selection. This article summarized the research progress of quantitative DNA analysis in oral carcinoma and precancerous lesions in recent years.

MicroRNA-155 in oral squamous cell carcinoma: Overexpression, localization, and prognostic potential.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is a common malignancy with poor prognosis. MicroRNAs (miRNAs) play an important role in cancer, but their role in OSCC is not clarified. METHODS: We performed miRNA microarray, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and fluorescence in situ hybridization (FISH) to examine miRNA expression in OSCC and paired adjacent cancer-free mucosal (ACF) tissues. RESULTS: Thirteen miRNAs, including miRNA-155, were upregulated (>2-fold) in OSCC against ACF. MiRNA-155 was confirmed to have significantly higher expression in OSCC against ACF (paired-samples t test; p = .041) and it was localized in the cancer nest, inflammatory area, and vascular endothelium of OSCC. High expression of miRNA-155 in ACF tissue was an independent prognostic indicator for OSCC survival. CONCLUSION: MiRNA-155 was overexpressed in OSCC and it was located in the cancer nest, inflammatory area, and vascular endothelium of OSCC. High miRNA-155 expression level in ACF may predict poor prognosis in patients with OSCC.

[The effect of retrovirus-mediated hTRT transfection into cultured oral keratinocytes].

PURPOSE: Human telomerase reverse transcriptase (hTRT) was transfected into cultured oral keratinocytes (OKC) mediated by pBABE-tert recombined retrovirus to investigate the effect on OKC lifespan. METHODS: pBABE-tert recombined retrovirus loaded with hTRT gene was amplified by transfected PT67 cells, and then transfected into cultured OKC in vitro. The positive clones of OKC were separated by puromycin and subcultured. Telomerase activity was analyzed by telomerase PCR-ELISA and PCR-PAGE. RESULTS: The hTRT positive clones of OKC showed telomerase expression, with extending lifespan to 8-9 passages. CONCLUSIONS: The hTRT transfected OKC can prolong doubly lifespan but not be immortalized, which indicates that cellular immortality mechanism is complicated and multi-controled. Telomerase activity is the key for cell immortalization but not the only impact factor.

DAPK promoter hypermethylation in tissues and body fluids of oral precancer patients.

Death-associated protein kinase (DAPK) has been suggested as a tumor suppressor gene. A high frequency of DAPK promoter hypermethylation has been noted in head and neck cancers and other solid tumors, and it has been used as a tumor marker in molecular detection strategies. Our aim was to examine DAPK promoter hypermethylation in tissue, blood, and salivary rinse samples of oral precancer patients (OPs) and to explore the potential role in oral carcinogenesis. DAPK hypermethylation was analyzed in 77 OPs and 32 oral squamous cell carcinomas (OSCCs) by real-time quantitative methylation-specific PCR (QMSP). We compared the hypermethylation expression between two groups and analyzed the associations with clinicopathologic parameters. The promoter hypermethylation frequency of DAPK in tissue (46.9%) and blood (52.2%) of OSCCs was significantly higher than those in OPs (19.5%, P = 0.004; 22.4%, P = 0.007, respectively). DAPK promoter hypermethylation expression in blood was correlated with its expression in tissue (r = 0.49, P < 0.000). The OP patients who smoked more than 20 years were found 40.0% tissue DAPK hypermethylation in contrast with 10.7% tissue DAPK hypermethylation in the patients whose smoking duration ≦20 years (P = 0.010). Our results suggest that DAPK hypermethylation is an early event in oral carcinogenesis and blood DAPK hypermethylation might be a potential minimal invasive biomarker for OSCC early detection.