Thirteen-gene DNA methylation analysis of oral brushing samples: A potential surveillance tool for periodic monitoring of treated patients with oral cancer.

BACKGROUND: We evaluated the prognostic role of 13-gene DNA methylation analysis by oral brushing repeatedly performed during the follow-up of patients surgically treated for oral cancer. METHODS: This is a nested case-control study including 61 patients for a total of 64 outcomes (2/61 patients experienced multiple relapses). Samples were collected at baseline (4-10 months after OSCC resection) and repeatedly every 4-10 months until relapse or death. DNA methylation scores were classified as persistently positive, persistently negative, or mixed. RESULTS: Twenty cases who had persistently positive scores and 30 cases with mixed scores had, respectively, an almost 42-fold (p < 0.001) and 32-fold (p = 0.006) higher likelihood of relapse, compared to 14 patients with persistently negative scores. The last score before reoccurrence was positive in 18/19 secondary events. CONCLUSIONS: The 13-gene DNA methylation analysis may be considered for the surveillance of patients treated for oral carcinoma.

Topical applications of heterologous platelet rich plasma (PRP) for refractory gingival lesions in autoimmune blistering diseases.

BACKGROUND: Recalcitrant gingival erosions, blisters and desquamative gingivitis are common features in oral autoimmune blistering diseases (AIBD). First line treatments include high-dosage corticosteroids and other immunosuppressive drugs, with several side effects and elevated number of recurrences. Autologous platelet-rich plasma (PRP) has been recently introduced as an alternative treatment and its use seems to be promising and safe. METHODS: In this study we describe the use of topical application of heterologous PRP in nine patients affected by mucous membrane pemphigoid, with gingival lesions refractory to previous treatments. Topical applications of PRP were performed once a week for 2 months and the endpoint for clinical evaluation was set 3 months after the last session. Oral disease severity score (ODSS) and VAS scores for pain measurement were recorded before and after treatment. RESULTS: The procedure was painless, well accepted, and free from adverse reactions. All patients (100%) reported a reduction in VAS whereas reduction in ODSS was observed in 89% of patients. CONCLUSIONS: Within the limits of the study, topical heterologous PRP is a safe and promising procedure to be studied in future controlled randomized trials as adjuvant treatment for refractory gingival lesions in patients with AIBDs.

Pre-operative incisional biopsy of oral squamous cell carcinoma: high podoplanin expression is related to perineural invasion and may be a useful predictor of disease progression.

OBJECTIVE: Immunohistochemical analysis of podoplanin expression as a pre-operative molecular marker for perineural invasion (PNI) may represent an attractive strategy for surgical management of oral squamous cell cancer (OSCC). We evaluated the relationship between podoplanin expression and PNI in pre-operative incisional biopsies of OSCC. STUDY DESIGN: After performing pathological staging and histologic and immunohistochemical evaluation of 83 surgical specimens, we performed multivariable logistic regression analysis to examine the relationship between PNI and independent variables. To evaluate the utility of podoplanin immunopositivity for discrimination of PNI status pre-operatively, we calculated the sensitivity, specificity, positive predictive value, and negative predictive value. We performed receiver operating characteristic curve analysis to evaluate the diagnostic accuracy of podoplanin immunopositivity for predicting PNI alone and in combination with age, T stage, N stage, and index site. RESULTS: We observed podoplanin expression in 42 (50.6%) of all the 83 pre-operative incisional biopsies and 29 of the pre-operative biopsies of the 31 (93.5%) postoperative specimens with PNI. The rate of podoplanin expression was significantly higher in patients with pT3 to pT4 stage and pN+ stage disease. Podoplanin positivity in the pre-operative biopsy showed high sensitivity in predicting PNI in the surgical specimen. CONCLUSION: Podoplanin expression appears to be an independent pre-operative variable significantly related to PNI and a possibly valuable prognostic marker for therapeutical planning and surgical treatment of OSCC.

Patient-reported outcome measures after periodontal surgery.

OBJECTIVES: The present study aimed to explore the impact of different periodontal surgical treatments on the quality of life and postoperative morbidity. MATERIALS AND METHODS: The present study is a single-center, prospective, observational cohort trial. One hundred fifty-five patients, referred to the Periodontal Department of Bologna University who needed periodontal surgical treatment, were recruited. The self-reported perception of the postoperative course was assessed using the following anonymous questionnaires: Italian oral health impact profile (I-OHIP-14), visual analog scale (VAS) to evaluate the intensity of the pain, and 5-point Likert scale. RESULTS: Patients reported a mean OHIP-14 total score of 9.87±8.5 (range 0-42), significantly influenced by the female sex, flap extension, and periodontal dressing. A mean VAS score of 2.96±2.39 (range 0-9) was calculated, and was found to be influenced by the presence of vertical releasing incisions and palatal flap extension. Of the 155 subjects, 40 (25.8%) patients reported bleeding as a post-surgical complication, 96 (61.9%) swelling, 105 (67.7%) eating discomfort, and 44 (28.4%) reported speech discomfort. CONCLUSIONS: Within the limitations of the nature of the present study, periodontal surgical procedures have a low impact on patients' quality of life evaluated through the OHIP-14 and VAS pain questionnaires. CLINICAL RELEVANCE: Periodontal surgical procedures are safe procedures, with a limited duration of postoperative discomfort as well as the incidence of complications.

Shared epigenetic alterations between oral cancer and periodontitis: A preliminary study.

INTRODUCTION: We recently developed a non-invasive sampling procedure for oral squamous cell carcinoma (OSCC) detection based on DNA methylation analysis of a panel of 13 genes. Oral cancer, as well as acute and chronic inflammatory diseases, may influence the methylation level of several genes in the oral cavity. In the present study, we evaluated the presence of periodontal disease (PD) and the methylation status using our 13-gene panel. METHODS: Oral brushing specimens were collected from three different patient groups: 23 gingival OSCC patients, 15 patients affected by PD, and 15 healthy volunteers lacking evidence of PD. DNA methylation analysis was performed and each sample was determined to be positive or negative based on a predefined cut-off value. RESULTS: Positive results were found for 23/23 OSCC patients, 3/15 PD patients, and 0/15 samples from healthy volunteers. The GP1BB and MIR193 genes in the PD group exhibited mean methylation levels similar to OSCC patients. ZAP70 showed different methylation levels among three groups. CONCLUSION: Preliminary data identified shared epigenetic alterations between PD and OSCC patients in two inflammatory genes (GP1BB and MIR193). This study may help to identify potential links between the two diseases and serve as a starting point for the future research focused on pathogenesis.

A 13-Gene DNA Methylation Analysis Using Oral Brushing Specimens as an Indicator of Oral Cancer Risk: A Descriptive Case Report.

Analysis of genetic or epigenetic markers from saliva or brushing specimens has been proposed as a diagnostic aid to identify patients at risk of developing oral cancer. However, no reliable non-invasive molecular method for this purpose is commercially available. In the present report, we describe the potential application of a procedure based on a 13-gene DNA methylation analysis using oral brushing samples from a patient affected by oral leukoplakia who developed two metachronous oral carcinomas during the follow-up period. A positive or a negative score was calculated for each brushing sample based on a predefined cut-off value. In this patient, a positive score was detected in the oral leukoplakia diagnosed more than 2 years before the development of oral squamous cell carcinoma and subsequently in clinically healthy mucosa 8 months before the appearance of a secondary tumor. This suggests a potential role of our procedure as an indicator of oral cancer risk.

Clinical validation of 13-gene DNA methylation analysis in oral brushing samples for detection of oral carcinoma: Italian multicenter study.

BACKGROUND: The aim of this Italian multicenter study was to evaluate the diagnostic performance of a minimally invasive method for the detection of oral squamous cell carcinoma (OSCC) based on 13-gene DNA methylation analysis in oral brushing samples. METHODS: Oral brushing specimens were collected in 11 oral medicine centers across Italy. Twenty brushing specimens were collected by each center, 10 from patients with OSCC, and 10 from healthy volunteers. DNA methylation analysis was performed in blindness, and each sample was determined as positive or negative based on a predefined cutoff value. RESULTS: DNA amplification failed in 4 of 220 (1.8%) samples. Of the specimens derived from patients with OSCC, 93.6% (103/110) were detected as positive, and 84.9% (90/106) of the samples from healthy volunteers were negative. CONCLUSION: These data confirmed the diagnostic performance of our novel procedure in a large cohort of brushing specimens collected from 11 different centers and analyzed in blindness.

Pre-Operative Evaluation of DNA Methylation Profile in Oral Squamous Cell Carcinoma Can Predict Tumor Aggressive Potential.

BACKGROUND: Prognosis of oral squamous cell carcinoma (OSCC) is difficult to exactly assess on pre-operative biopsies. Since OSCC DNA methylation profile has proved to be a useful pre-operative diagnostic tool, the aim of the present study was to evaluate the prognostic impact of DNA methylation profile to discriminate OSCC with high and low aggressive potential. METHODS: 36 OSCC cases underwent neoplastic cells collection by gentle brushing of the lesion, before performing a pre-operative biopsy. The CpG islands methylation status of 13 gene (ZAP70, ITGA4, KIF1A, PARP15, EPHX3, NTM, LRRTM1, FLI1, MiR193, LINC00599, MiR296, TERT, GP1BB) was studied by bisulfite Next Generation Sequencing (NGS). A Cox proportional hazards model via likelihood-based component-wise boosting was used to evaluate the prognostic power of the CpG sites. RESULTS: The boosting estimation identified five CpGs with prognostic significance: EPHX3-24, EPHX3-26, ITGA4-3, ITGA4-4, and MiR193-3. The combination of significant CpGs provided promising results for adverse events prediction (Brier score = 0.080, C-index = 0.802 and AUC = 0.850). ITGA4 had a strong prognostic power in patients with early OSCC. CONCLUSIONS: These data confirm that the study of methylation profile provides new insights into the molecular mechanisms of OSCC and can allow a better OSCC prognostic stratification even before surgery.

An Evolutionary Cancer Epigenetic Approach Revealed DNA Hypermethylation of Ultra-Conserved Non-Coding Elements in Squamous Cell Carcinoma of Different Mammalian Species.

BACKGROUND: Ultra-conserved non-coding elements (UCNEs) are genomic sequences that exhibit > 95% sequence identity between humans, mammals, birds, reptiles, and fish. Recent findings reported their functional role in cancer. The aim of this study was to evaluate the DNA methylation modifications of UNCEs in squamous cell carcinoma (SCC) from different mammal species. METHODS: Fifty SCCs from 26 humans, 17 cats, 3 dogs, 1 horse, 1 bovine, 1 badger, and 1 porcupine were investigated. Fourteen feline stomatitis and normal samples from 36 healthy human donors, 7 cats, 5 dogs, 5 horses, 2 bovines and 1 badger were collected as normal controls. Bisulfite next generation sequencing evaluated the DNA methylation level from seven UCNEs (uc.160, uc.283, uc.416, uc.339, uc.270, uc.299, and uc.328). RESULTS: 57/59 CpGs were significantly different according to the Kruskal-Wallis test (p < 0.05) comparing normal samples with SCC. A common DNA hypermethylation pattern was observed in SCCs from all the species evaluated in this study, with an increasing trend of hypermethylation starting from normal mucosa, through stomatitis to SCC. CONCLUSIONS: Our findings indicate that UCNEs are hypermethylated in human SCC, and this behavior is also conserved among different species of mammals.

Application of a non-invasive oral brushing procedure based on bisulfite sequencing of a 13-gene panel to study high-risk OSCC patients.

BACKGROUND: A non-invasive sampling procedure for the early detection of Oral Squamous Cell Carcinoma (OSCC) based on DNA methylation analysis of a panel of 13 genes was applied in 4 different OSCC risk-group of patients. Aim of the study is to evaluate the between-group differences and the variables related to the methylation profile of each group. METHODS: Oral brushing samples were collected from 54 healthy subjects, 31 Oral Leukoplakia (OL) patients, 18 Oral Lichen Planus (OLP) patients and 26 patients previously treated for OSCC. Each sample was considered positive or negative in relation to a predefined cut-off value. RESULTS: None of the samples from 54 healthy subjects were positive, whereas 22/31 OL, 3/18 OLP and 8/26 surgically treated OSCC samples showed positive values with respect to the cut-off. In OL patients, dysplasia was the only variable significantly related to positive values: 10/10 OLs with high-grade dysplasia were positive with respect to 12/21 OLs without dysplasia (Chi 6.039, p< 0.05). CONCLUSION: DNA methylation analysis in epithelial cells collected by oral brushing seems to be a promising genetic method to distinguish lesions at high risk of developing OSCC. Larger population studies and an adequate follow-up period are necessary to confirm these preliminary data.

13-gene DNA Methylation Analysis from Oral Brushing: A Promising Non Invasive Tool in the Follow-up of Oral Cancer Patients.

BACKGROUND: This study aimed to evaluate the prognostic value of a non-invasive sampling procedure based on 13-gene DNA methylation analysis in the follow-up of patients previously treated for oral squamous cell carcinoma (OSCC). METHODS: The study population included 49 consecutive patients treated for OSCC. Oral brushing sample collection was performed at two different times: before any cancer treatment in the tumor mass and during patient follow-up almost 6 months after OSCC treatment, within the regenerative area after OSCC resection. Each sample was considered positive or negative in relation to a predefined cut-off value. RESULTS: Before any cancer treatment, 47/49 specimens exceeded the score and were considered as positive. Six months after OSCC resection, 16/49 specimens also had positive scores in the samples collected from the regenerative area. During the follow-up period, 7/49 patients developed locoregional relapse: 6/7 patients had a positive score in the regenerative area after OSCC resection. The presence of a positive score after oral cancer treatment was the most powerful variable related to the appearance of locoregional relapse. CONCLUSION: 13-gene DNA methylation analysis by oral brushing may have a clinical application as a prognostic non-invasive tool in the follow-up of patients surgically treated for OSCC.

A Noninvasive Test for MicroRNA Expression in Oral Squamous Cell Carcinoma.

MicroRNAs have recently been proposed as non-invasive biomarkers in Oral Squamous Cell Carcinoma (OSCC). The aim of this study was to analyze the expression of a panel of miRNAs in epithelial cells collected by oral brushing from OSCCs from regenerative areas after OSCC surgical resection and from their respective normal distant mucosa. Oral brushing specimens were collected from 24 healthy donors, 14 OSCC patients with specimens from tumour and normal distant mucosa, and from 13 patients who had OSCC resection, with samples from regenerative areas after OSCC resection and normal distant mucosa. Expression levels of eight targets (miR-21, miR-375, miR-345, miR-181b, miR-146a, miR-649, miR-518b, and miR-191) were evaluated by real-time Polymerase Chain Reaction (PCR). A highly significant between-group difference was found for miR-21 (F = 6.58, p < 0.001), miR-146a (F = 6.974, p < 0.001), and miR-191 (F = 17.07, p < 0.001). The major difference was observed between samples from healthy donors and from OSCC brushing, whereas no significant differences were observed between areas infiltrated by OSCC and their respective normal distant mucosa. Furthermore, altered expression of miR-146a and miR-191 was also observed in regenerative areas after OSCC resection. CONCLUSIONS: Oral brushing could be proposed as a noninvasive method to study microRNA expression in oral mucosa in OSCC patients.

Predictive Role of p53 Protein as a Single Marker or Associated with ki67 Antigen in Oral Leukoplakia: A Retrospective Longitudinal Study.

Oral leukoplakia (OL) is the most common potentially malignant lesion of the oral cavity. Immunohistochemical analysis of p53 and Ki67 proteins is a simple and inexpensive method widely used in non-dysplastic OLs to reveal lesions predicted to develop oral cancer. The present longitudinal study evaluated the predictive role of p53 and Ki67 proteins alone or in combination in a group of OLs without dysplasia followed for many years. Seventy-seven OL patients referred to our Department between January 2006 and October 2013 underwent histochemical analysis of p53 and Ki67 expression. OLs were considered at high risk in the presence of either high p53 expression (>20%), or low/normal p53 expression associated with high Ki67 expression (Ki67/p53 ratio >3). Seven OLs evolved to OSCC during the follow-up period. Three cases had p53 overexpression, while four had a high Ki67/p53 ratio. Statistical significance was reached when samples with p53 overexpression were combined with samples with high Ki67/p53 ratio (Chi square 5.3; p<0.02). The combined immunohistochemical expression of p53 and Ki67 proteins could be a useful and simple molecular marker for early detection of non-dysplastic OLs at risk of developing oral cancer.

Paraesthesia after local anaesthetics: an analysis of reports to the FDA Adverse Event Reporting System.

This study was aimed to evaluate the possible alert signals of paraesthesia by local anaesthetics, focusing on those used in dentistry. A case/non-case study of spontaneous adverse events recorded in FAERS (FDA Adverse Event Reporting System) between 2004 and 2011 was performed. Cases were represented by the reports of reactions grouped under the term 'Paraesthesias and dysaesthesias' involving local anaesthetics (ATC: N01B*); non-cases were all other reports of the same drugs. Reporting odds ratios (ROR) with the relevant 95% confidence intervals (95CI) were calculated. Alert signal was considered when number of cases >3 and lower limit of ROR 95CI > 1. To estimate the specificity of signals for dentistry, the analysis was restricted to the specific term "Oral Paraesthesia" and to reports concerning dental practice. Overall, 528 reports of 'Paraesthesias and dysaesthesias' were retrieved, corresponding to 573 drug-reaction pairs (247 lidocaine, 99 bupivacaine, 85 articaine, 30 prilocaine, 112 others). The signal was significant only for articaine (ROR=18.38; 95CI = 13.95-24.21) and prilocaine (2.66; 1.82-3.90). The analysis of the specific term "Oral Paraesthesia" retrieved 82 reports corresponding to 90 drug-reaction pairs (37 articaine, 19 lidocaine, 14 prilocaine, 7 bupivacaine, 13 others) and confirmed the signal for articaine (58.77; 37.82-91.31) and prilocaine (8.73; 4.89-15.57). The analysis of reports concerning dental procedures retrieved a signal for articaine, both for any procedures (8.84; 2.79-27.97) and for non-surgical ones (15.79; 1.87-133.46). In conclusion, among local anaesthetics, only articaine and prilocaine generated a signal of paraesthesia, especially when used in dentistry.

p16(INK4) expression is not associated with human papillomavirus in oral lichen planus.

OBJECTIVE: Frequencies as high as 60% of overexpressed p16(INK4) were recently reported in lichen planus (LP). Because p16(INK4) overexpression may be a feature of human papilloma virus (HPV)-induced cancer, it has been postulated that LP may be somehow related to HPV. The present study is the first to evaluate both high p16(INK4) expression and HPV in patients with LP. STUDY DESIGN: Thirty-five consecutive biopsy specimens from patients with LP constituted the basis of the present study. Level of p16(INK4A) expression was evaluated in each sample by immunohistochemical analysis, and the presence of HPV DNA was tested by real-time polymerase chain reaction (PCR). RESULTS: p16(INK4) expression was detected in 26 specimens, whereas HPV was found in 4 lesions: 3 low-risk HPV and 1 high-risk HPV. All HPV-positive lesions also indicated p16(INK4A) overexpression, whereas 22 cases of overexpressed p16(INK4A) were HPV negative (Chi square 2.6; ns). CONCLUSIONS: p16(INK4) overexpression is not correlated with HPV in patients with LP.

Clinical and histologic healing of lichenoid oral lesions following amalgam removal: a prospective study.

OBJECTIVE: This study aimed to see whether clinical healing after amalgam removal corresponds to histologic healing, i.e., a complete disappearance of any histologic sign of lichenoid lesion. STUDY DESIGN: The study evaluated 64 patients with lichenoid lesions and at least one amalgam filling. RESULTS: After amalgam removal, complete clinical healing was obtained in 14 patients (22%) and was significantly related to lesion topography (χ(2) 4.7; P < .05) and positive patch test (χ(2) 6.3; P < .01). Complete histologic healing was obtained in only 7 cases (50% of clinically healed patients), and was significantly related to the combination of positive patch test and strict contact with amalgams (Fisher's exact test P < .01). CONCLUSIONS: Contact with amalgams and positive patch testing are good but not absolute indicators of the beneficial effect of amalgam replacement. In addition, complete clinical healing does not necessarily mean a disappearance of the histologic characteristics of OLL/OLP lesions.

Ki-67 from clinically and histologically "normal" distant mucosa as prognostic marker in early-stage (T1-T2N0) oral squamous cell carcinoma: a prospective study.

PURPOSE: The aggressive behavior and long-term prognosis of oral squamous cell carcinoma (OSCC) have recently been related to the mucosa surrounding the primary mass, consisting of genetically altered cells that might be responsible for cancer progression. Early-stage T1-T2N0 OSCCs have been associated with a good prognosis; however, a certain percentage of them can be complicated by locoregional metastases. The purpose of our study was to determine whether an abnormal proliferative status can be found in clinically and histologically "normal" mucosa situated in areas distant from the primary tumor. We also sought to determine whether this is associated with a poor prognosis in terms of local recurrence or lymph node metastasis. PATIENTS AND METHODS: The prospective study included 42 consecutive patients with T1N0M0 (n = 19) and T2N0M0 (n = 23) OSCC. Disease-free survival endpoints were defined as the duration between surgical resection and the diagnosis of recurrence, lymph node metastasis, or last follow-up visit. Proliferative status in distant areas (opposite cheek) was evaluated by Ki-67 expression. RESULTS: The mean Ki-67 value (17.6% ± 8.2%) in the distant mucosa was significantly greater (F = 13.87; P < .01) than that found in the controls (9.8 ± 3.1). "Abnormally high" Ki-67 values were detected in 13 patients with OSCC (30%). Four patients developed locoregional recurrence during follow-up. Kaplan-Meier analysis showed that Ki-67 in the distant mucosa was a significant independent prognostic factor for disease-free survival. CONCLUSIONS: A certain percentage of patients surgically treated for early T1-T2 OSCC will have an abnormal proliferative status in areas very distant from the primary tumor that seems to be related to a poor prognosis.

Cancerization of cutaneous flap reconstruction for oral squamous cell carcinoma: report of three cases studied with the mtDNA D-loop sequence analysis.

AIMS: Tissue defects, resulting from surgical resection of oral squamous cell carcinoma (OSCC), are reconstructed routinely with skin grafts. OSCC arising from the grafted skin has been described; however, it is still unclear whether primary and second tumours have a common clonal origin. The aim of this study was to evaluate the clonal relationship between the primary OSCC and secondary neoplastic changes appearing in the skin graft in three patients, by screening the mitochondrial DNA D-loop region (mtDNA). METHODS AND RESULTS: In all three cases, the neoplastic lesions arising in the skin graft showed a clonal relationship with the previous OSCC and, on the basis of the results obtained by mtDNA analysis, could be considered to be a recurrence of the primary OSCC rather than a second primary OSCC. CONCLUSIONS: Starting from a field of genetically altered cells in the oral mucosa, the spread of the clonal cell population to the cutaneous flap might be stimulated by cytokines produced by the grafted skin. More studies are needed to evaluate the molecular relationship between primary and second OSCC to identify patients at higher risk of developing a second tumour in the skin graft.