Improvement in the risk assessment of oral leukoplakia through morphology-related copy number analysis.

Oral leukoplakia is the most common type of oral potentially malignant disorders and considered a precursor lesion to oral squamous cell carcinoma. However, a predictor of oral leukoplakia prognosis has not yet been identified. We investigated whether copy number alteration patterns may effectively predict the prognostic outcomes of oral leukoplakia using routinely processed paraffin sections. Comparison of copy number alteration patterns between oral leukoplakia with hyperplasia (HOL, n=22) and dysplasia (DOL, n=21) showed that oral leukoplakia with dysplasia had a higher copy number alteration rate (86%) than oral leukoplakia with hyperplasia (46%). Oral leukoplakia with dysplasia exhibited a wider range of genomic variations across all chromosomes compared with oral leukoplakia with hyperplasia. We also examined a retrospective cohort of 477 patients with oral leukoplakia with hyperplasia with detailed follow-up information. The malignant transformation (MT, n=19) and leukoplakia recurrence (LR, n=253) groups had higher frequencies of aneuploidy events and copy number loss rate than the free of disease (FD, n=205) group. Together, our results revealed the association between the degree of copy number alterations and the histological grade of oral leukoplakia and demonstrated that copy number alteration may be effective for prognosis prediction in oral leukoplakia patients with hyperplasia.

Prognostic Implications of DNA Repair, Ploidy and Telomerase in the Malignant Transformation Risk Assessment of Leukoplakia.

BACKGROUND: Although leukoplakia shows a higher risk for malignant transformation to oral cancer, currently there are no clinically relevant biomarker which can predict the potentially high risk leukoplakia. This study aimed to investigate the genetic alterations such as DNA ploidy, telomerase expression and DNA repair capacity as predictive markers of malignant transformation risk of leukoplakia. METHODS: The study was initiated in September 2005 and patients were followed up to March 2014. Two hundred patients with oral leukoplakia, 100 patients with oral cancer and 100 healthy, age and sex matched adults with normal oral mucosa as controls were recruited. The DNA ploidy content was measured by high resolution flow cytometry, level of telomerase expression was identified by TRAP assay and intrinsic DNA repair capacity was measured by mutagen induced chromosome sensitivity assay of cultured peripheral blood lymphocytes. The Chi-square test or Fisher's Exact test was used for comparison of categorical variables between biomarkers. A p value less than or equal to 0.05 was considered as statistically significant. Analysis was performed with SPSS software version 16. Logistic regression was used to find the association between the dependent and three independent variables. RESULTS: There was significant difference in the distribution of ploidy status, telomerase activity and DNA repair capacity among control, leukoplakia and oral cancer group (p<0.001). When the molecular markers were compared with histological grading of leukoplakia, both DNA ploidy analysis and telomerase activity showed statistical significance (p<0.001). Both aneuploidy and telomerase positivity was found to coincide with high-risk sites of leukoplakia and were statistically significant (p.

Diagnostic Utility of Cytology in Assessment of Ploidy Status in Potentially Malignant Oral Disorders.

INTRODUCTION: Oral leukoplakia, the most common potentially malignant oral disorder (PMOD) may progress to oral squamous cell carcinoma (OSCC). Although, the current standard of care for assessing its malignant potential remains histological examination and assessing the severity of dysplasia, DNA ploidy analysis has been suggested as a surrogate marker to predict the behaviour of PMODs. OBJECTIVES: To detect aneuploidy and to correlate ploidy status with different grades of dysplasia in both tissue and cytology samples to predict the behaviour of these potentially malignant disorders and to assess the diagnostic utility of cytology samples for ploidy analysis. METHODOLOGY: After obtaining ethical clearance and consent, tissue and cytology samples of leukoplakia were collected and grouped based on the dysplastic findings into low-risk (n=20) and high-risk (n=20). DNA ploidy analysis was done using high resolution flow cytometry and its diagnostic utility was assessed. RESULTS: Diagnostic utility was expressed in terms of sensitivity, specificity, PPV and NPV. On comparing the ploidy status of individual cases between tissue and cytology samples, cytology was able to accurately determine the ploidy status in majority of the cases. In the low-risk group, cytology had a sensitivity and specificity of 100% and a PPV and NPV of 100% with an overall diagnostic accuracy of 100%. Among the high-risk group, cytology had a sensitivity of 80% and specificity of 100% with a PPV of 100% and NPV of 83.33% and had an overall diagnostic accuracy of 90%. Combining both groups together, it had a sensitivity of 85.71% and specificity of 100% with a PPV of 100% and NPV of 92.31% and had an overall diagnostic accuracy of 94.74%. CONCLUSION: Overall, this study showed a positive correlation between cytology and tissue samples and ploidy and grade of dysplasia and cytology proved to be a simple and efficient with a reasonable diagnostic value.

Assessment of DNA Damage in Leukoplakia Patients with Different Degrees of Dysplasia.

BACKGROUND: Single cell gel electrophoresis (SCGE) assay also known as comet assay is a rapid and highly sensitive fluorescent molecular technique for detecting various forms of deoxyribonucleic acid (DNA) damage at individual cellular level. MATERIALS AND METHODS: The present study was done to detect the extent of DNA damage in oral leukoplakia (OL) and compare with normal individuals. The sample population was obtained from an outpatient clinic of a tertiary teaching dental institute. A total of 36 consecutive patients with leukoplakia and 10 healthy normal volunteers were recruited for the study and assessed for the extent of DNA damage using SCGE following clinical diagnosis and histological grading. Peripheral blood was obtained by venipuncture and SCGE assay was performed. Mean comet tail length was recorded and analyzed statistically to compare the extent of damage in each group. RESULTS: The mean comet tail length seen in leukoplakia patients with moderate to severe dysplasia was 1.25 ± 0.14 mm while for the control subjects, it was 0.31 ± 0.10 mm. The difference was statistically significant (p = 0.000). On comparing within the grades of leukoplakia, a progressive trend of increasing tail length was observed with increasing grades of dysplasia. CONCLUSION: Deoxyribonucleic acid damage as measured by SCGE is seen in leukoplakia. A stepwise increase in DNA damage levels from healthy controls, through patients with non-dysplastic epithelium to varying grades of dysplasia has been observed indicating the extent of DNA damage in this high risk group.

Progress risk assessment of oral premalignant lesions with saliva miRNA analysis.

BACKGROUND: Oral cancer develops through multi-stages: from normal to mild (low grade) dysplasia (LGD), moderate dysplasia, and severe (high grade) dysplasia (HGD), to carcinoma in situ (CIS) and finally invasive oral squamous cell carcinomas (OSCC). Clinical and histological assessments are not reliable in predicting which precursor lesions will progress. The aim of this study was to assess the potential of a noninvasive approach to assess progress risk of oral precancerous lesions. METHODS: We first used microRNA microarray to profile progressing LGD oral premaligant lesions (OPLs) from non-progressing LGD OPLs in order to explore the possible microRNAs deregulated in low grade OPLs which later progressed to HGD or OSCC. We then used RT-qPCR to detect miRNA targets from the microarray results in saliva samples of these patients. RESULTS: We identified a specific miRNA signature that is aberrantly expressed in progressing oral LGD leukoplakias. Similar expression patterns were detected in saliva samples from these patients. CONCLUSIONS: These results show promise for using saliva miRNA signature for monitoring of cancer precursor lesions and early detection of disease progression.

Frequent microsatellite alterations at chromosomes 9p21 and 3p14 in oral premalignant lesions and their value in cancer risk assessment.

To better understand genetic alterations in oral premalignant lesions, we examined 84 oral leukoplakia samples from 37 patients who had been enrolled in a chemoprevention trial. The samples were analyzed for two microsatellite markers located at chromosomes 9p21 and 3p14. Loss of heterozygosity (LOH) at either or both loci was identified in 19 of the 37 (51%) patients. Of these 19 patients, seven (37%) have developed head and neck squamous cell carcinoma (HNSCC) while only one of 18 (6%) of patients without LOH developed HNSCC. Our data suggest that clonal genetic alterations are common in oral premalignant lesions; that multiple genetic alterations have already occurred in oral premalignant lesions, allowing at least a focal clonal expansion; and that losses of the 9p21 and 3p14 regions may be related to early processes of tumorigenesis in HNSCC. These genetic alterations in premalignant tissues may serve as markers for cancer risk assessment.