Melbourne Dental School - Theses
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ItemOral cancer prediction using microRNA in oral swirls( 2018)Purpose: To study a clinically convenient sample type, oral swirls as a source of microRNA for analysis in oral disease states. Objectives: To study a panel of OSCC-associated microRNA, identified in next generation sequencing (NGS) data of tissue specimens, in oral swirls from individuals with OSCC and oral potentially malignant disorders (OPMDs). Materials and Methods: Oral swirls were inspected using electron microscopy and tested for robustness by challenge with RNase and temperature shifts and analysis using microRNA by qPCR. Oral swirls were collected from 190 individuals with and without oral mucosal conditions. An OSCC- associated panel of microRNAs was identified in FFPE specimen NGS data and a fresh frozen specimen data set from The Cancer Genome Atlas. This panel was studied by qPCR in the oral swirls from 190 individuals with and without mucosal abnormalities including OSCC (n=53) and OPMDs (n=74). Results: Oral swirl sourced microRNA was consistently detected and demographics, comorbidities and oral disease states did not affect the yield of RNA. A reproducible workflow was used to extract RNA from oral swirls collected from 190 individuals. Upregulation of miR-31, miR-21 and downregulation of miR-99a, let-7c, miR-125b and miR-100 was found between OSCC and controls in NGS data of both FFPE and fresh frozen specimens. These microRNAs were studied in a training set of 15 OSCC vs 15 control oral swirls to develop a cumulative dysregulation score (AUC 0.95 (95% CI, 0.88-1.03)) and categorical algorithm-determined risk category. Utilizing the presence of HIGH-risk in 53 OSCC vs 54 controls, the test was 86.8% sensitive and 81.5% specific. One case of malignant transformation within the OPMD cohort demonstrated longitudinal utility of the test. Conclusion: Oral swirls provide a clinically convenient sample type for assessment of microRNA in disease states. This is first study to analyze microRNA sourced from oral swirls from individuals with and without mucosal abnormalities including OSCC and OPMDs. A HIGH-risk dysregulation signature was found to be accurate in indicating the presence of OSCC and exampled to parallel malignant transformation. Assessment in further longitudinal studies is warranted.
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ItemDevelopment of a molecular tool for oral squamous cell carcinoma risk assessment using microrna markers( 2014)Purpose The aim was to develop a robust methodology for analysis of microRNAs by assessing the validity of using formalin fixed paraffin embedded (FFPE) tissues, determining the most optimal cDNA conversion method and establishing a sound methodology for data analysis using PCR threshold, microRNA efficiency and appropriate normalization techniques. In addition, the aim was also to develop a microRNA biomarker panel and develop an algorithm for assessment of oral squamous cell carcinoma (OSCC) risk using microRNA abundance data. And finally, to assess the clinical utility of microRNA in oral cytological scrapings. Material and Methods To assess the utility of FFPE in microRNA analysis, 4 oral lesions were divided in half and were flash frozen and FFPE. Additionally, differing amounts of RNA were used to compare and establish the most sensitive means of cDNA conversion. Quantitative reverse transcription PCR (qRT–PCR) was conducted on all samples and comparisions were done to establish PCR efficiencies for microRNAs and establish appropriate methods for assessing PCR thresholds and normalization techniques. qRT-PCR was conducted on RNA isolated from 40 samples of formalin fixed and paraffin embedded (FFPE) biopsy samples that were histologically assessed as OSCC or histologically normal epithelium (HNE), as well as additonal oral tissue samples of oral lichen planus (OLP), mild, moderate and severe dysplasia. A panel of 11 microRNAs considered as potential biomarkers of OSCC were assessed with obtained Cq values normalized and statistically analyzed using qBase PLUS (Biogazelle) software. The microRNAs were evaluated by assessing oral cytological scrapings stored in different media at varying temperatures over differing time frames. Results Results showed that FFPE could be used for microRNA analysis along with pre- amplification in addition to Megaplex reverse transcription priming being the most sensitive method for cDNA conversion. Robust techniques were established for determining PCR thresholds and microRNA efficiencies along with global mean normalisation being deemed as the most appropriate normalization technique. Four microRNAs showed statistically significant abundance differences between the 20 OSCC and 20 HNE. Subsequently, an algorithm was developed for assessment of OSCC-risk (the “miR-OSCC-risk”) that gave sensitivity and specificity calculations for the HNE and OSCC samples tested. The algorithm output indicated high (flagged as red), indeterminate (amber) or low (green) risk, that showed a sensitivity of 95%; specificity of 70%; positive predictive value of 76%; negative predictive value of 93%; and an accuracy of 82.5%. The abundances of the microRNAs in the panel and the predictive accuracy of the algorithm were tested further using other oral lesion samples. Oral cytological scrapings of normal epithelia were in the majority of instances determined to be of low risk by the algorithm whereas the oral mucosal lichen planus and dysplastic lesions were in the majority determined to be of medium to high risk and warranting further clinical and histological investigation. Despite varying storage media, storage conditions and time frames of storage of cytological scrapings, microRNAs could be obtained and measured using qRT-PCR. Conclusions The developed methodology and miR-OSCC-risk algorithm can be used with qRT-PCR microRNA abundance data to provide an indication of OSCC risk that has potentially significant clinical utility. The diagnostic procedure developed here could be of benefit in the assessment of oral lesions of unknown pathology by the targeting of lesions requiring biopsy.
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ItemCandida virulence attributes: exploring a link to oral mucosal carcinogenesis( 2014)The fungus Candida has been implicated in the aetiology of oral cancer; however, the exact carcinogenic mechanisms are still under investigation. A persuasive body of evidence suggests a direct causal role involving metabolism and production of carcinogenic compounds such as acetaldehyde. Recently, the hypothesis of microbial induced inflammation is gaining an increased interest and thought to play an important role in the multi-step process of carcinogenesis. Chronic inflammatory mediators, such as cytokines and growth factors, may act as a link between infection, innate immunity, inflammation and cancer through their tumour growth stimulatory effect. Inadequate pathogen elimination, prolonged inflammatory signalling, and defects in anti-inflammatory mechanisms can lead to chronic inflammation and subsequent release of cytokines and growth factors that benefit tumour development. Previous research has found a significant association between the virulence potential of pathogenic fungi, such as secreted aspartyl proteinases and the expression of inflammatory cytokines and chemokines. In addition, differences have been observed in Candida strains/biotypes colonizing premalignant/malignant and healthy oral mucosa. The present research had four major aims: (1) To study the major virulence attributes and biofilm antifungal susceptibility of medically important Candida species and strains (2) To develop a rapid, accurate and economic genetic method for identification and genotyping of medically relevant fungal pathogens using real time polymerase chain reaction-high resolution melting curve analysis (HRMA) (3) To assess the prevalence of Candida carriage and level of colonization in oral cancer and matched non-oral cancer patients (4) To investigate the major virulence attributes and ethanol-derived acetaldehyde production of Candida isolated from oral cancer patients and compare these with oral yeasts isolated from patients with no evidence of oral cancer. Results showed a significant variation in biofilm forming ability and hydrolytic enzyme production between different Candida species and strains, with high antifungal resistance observed for C. albicans bioflms in comparison to planktonic Candida cells. Furthermore, correlation analysis revealed a significant positive correlation between biofilm forming ability and hydrolytic enzymes production. The HRMA-based genetic identification method developed during this study was rapid, cheap and highly accurate in identifying all unknown Candida isolates and C. albicans genotypes. The melting curve profiles of internal transcribed spacer regions and transposable intron in 25S of ribosomal DNA were highly discriminative and showed consistency over replicates and repeated assays. It had the advantages of using a single primer set to identify all unknown isolates with high potential to distinguish clinical isolates not included in the reference strain list. In the present study, the frequencies of oral yeasts’ presence and high oral colonization were significantly higher in oral cancer than non-oral cancer patients (p=001; p=0.033, respectively). No significant difference in the isolation profile of Candida species was found between the two groups, except C. parapsilosis was more frequent in the non-oral cancer group. Differences were noticed in the incidence of C. albicans strains where significantly more C. albicans genotype-A was isolated from cancer patients and significantly more C. albicans genotype-B isolated from non-cancer patients. Multiple regression analyses showed significant association with cancer observed for alcohol drinking (OR=4.253; 95% CI=1.351, 13.386), Candida presence (OR=3.242; 95% CI=1.505, 6.984) and high oral colonization (OR=3.587; 95% CI=1.153, 11.162). Finally, statistical analysis demonstrated significantly enhanced biofilm forming ability, phosphoplipase, proteinase and ethanol-derived acetaldehyde production by Candida isolated from oral cancer patients compared with isolates from matched non-oral cancer patients. A significant association was found between Candida species and certain virulence properties with higher frequency of high biofilm mass, high biofilm growth activity, high phospholipase and esterase activity was observed in C. albicans species as compared with the non-albicans Candida species. Acetaldehyde production was higher in C. albicans than non-albicans Candida isolates, but did not reach the level of statistical significance. A significantly higher frequency of high proteinase activity was seen in the non-albicans Candida compared with C. albicans isolates. No significant association was found between the genotype of C. albicans and any of the investigated virulence factors other than significantly higher biofilm mass was obtained in C. albicans genotype A compared with genotype B. Correlation analysis revealed a significant positive correlation between both biofilm mass and growth activity and phospholipase enzyme production.