Melbourne Dental School - Theses
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ItemNon-smokers, Non-drinkers: Assessing surgical outcomes in a clinically distinct cohort of Oral Squamous Cell Carcinoma patients( 2019)Background Tobacco and alcohol intake are responsible for approximately 65-70% and 20-35% respectively of oral squamous cell carcinomas (SCC). Non-smoking, non-drinking (NSND) patients represent approximately 13-35% of the oral SCC population and are more likely to be young (Mean 20-35 years) or elderly (Over 70 years) females with a predilection for tongue, gingivae and lower lip sites. Although approximately 24% of head and neck cancers occur in patients over 70 years old, there are few published reports of oral SCC in elderly patients. This group appears to be characterized by a higher proportion of NSND females. Bone invasion by oral SCC necessitates jaw resection. Ideally, pre-operative imaging can be used to guide resection. The current rate of non-invaded mandible resections ranges between 20 and 100%. Even with free-flap reconstruction, segmental resection still results in cosmetic and functional deficits, donor site morbidity and significant physiological strain resulting in increased risk, prolonged recovery and need for rehabilitation. Decreased physiological reserve and multiple medical co-morbidities make complex surgery undesirable in an elderly population. Marginal resection aims to maintain bony continuity to avoid complex reconstruction. Objectives The objectives of this study were: 1. Examine differences in survival and clinical outcomes of elderly patients without traditional risk factors presenting with oral squamous cell carcinoma. 2. Determine the accuracy of computed tomography (CT) and magnetic resonance imaging (MRI) at identifying bone invasion in oral SCC. Materials & Methods Retrospective review of 287 consecutive patients divided into 2 treatment period cohorts treated for oral SCC between the 1st Jan 2007 and 31st Dec 2012.. Patients were classified as either smoker-drinkers (SD) or non-smoking, non-drinking (NSND). Only patients with oral sub-site primaries according to ICD-10 were included. Carcinomas of the lip, tonsil, base of tongue and oro-pharyngeal sub-sites were excluded. A subset of 109 patients who underwent mandibular resection were also reviewed for bone invasion. 83 of these patients had pre-operative CT imaging studies of diagnostic quality available for review and 72 underwent MRI which were compared to histological resection specimens. Results Of the study population (N=287), 24.4% were NSND and 9.75% were NSND elderly (older than 70 years) females. Disease specific survival at 5 years was significantly reduced when NSND elderly females were compared to all other patients (p <0.001) as well as age matched controls (p = 0.006). This effect was verified independently in each cohort. Bone invasion was detected in 44 out of 109 (40.4%) resection specimens. Bone invasion was identified on CT imaging in 31 out of 83 cases (37.3%) and by MRI in 35 out of 72 cases (48.6%). The sensitivity and specificity of CT for detecting bone invasion was 69.0% and 79.6% respectively. The sensitivity and specificity of MRI for detecting bone invasion was 87.1% and 80.5% respectively. Conclusions The results of this study suggest that NSND elderly females are a distinct patient population with poorer disease specific survival outcomes and that negative imaging studies should not preclude an oncologically safe bony resection if indicated on clinical grounds.
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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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ItemSquamous cell carcinoma involving the maxilla: a 20 year retrospective review( 2015)Background: Squamous cell carcinoma (SCC) may primarily involve the maxilla via tumours of maxillary sinus origin or via tumours arising from the hard palate or maxillary alveolus within the oral cavity. Both of these presentations represent a small subset of head and neck squamous cell carcinoma making SCC involving the maxilla a relatively uncommon disease. Aims: To examine our institution’s experience with SCC involving the maxilla and to compare the outcomes of patients with SCC involving the maxilla via the oral cavity with patients presenting with maxillary sinus SCC. In addition to this we evaluated our experiences regarding cervical metastases in patients with being treated for maxillary sinus SCC. Materials and methods: A retrospective review of patients with SCC involving the maxilla treated with curative intent via the Head and Neck Oncology stream at the Royal Melbourne Hospital between 1990 and 2010 was undertaken. Data collected was analysed to determine the influence of primary site on patient outcomes. Data was also examined to determine the incidence of cervical metastases at presentation and subsequent regional failure in patients with maxillary sinus SCC. Results: Influence of site of origin on the outcome of squamous cell carcinoma involving the maxilla – oral versus sinus. When compared with tumours involving the maxilla via the oral cavity, maxillary sinus origin did not per se confer a poorer prognosis. Maxillary sinus tumours were however more likely to present with advanced T-stage (T3/T4) and were more likely to have positive margins after resection. Cervical metastases in maxillary sinus squamous cell carcinoma The incidence of clinically or radiographically detected cervical metastases in our patients was 11%. At our institution, the majority of patients treated for maxillary sinus SCC underwent elective neck irradiation, with an overall regional failure rate of 4%. Our results found that local recurrence rather than regional metastasis was the primary source of treatment failure Conclusions: Influence of site of origin on the outcome of squamous cell carcinoma involving the maxilla – oral versus sinus. Site of origin does not in of itself confer a poorer prognosis; rather maxillary sinus SCC is more likely to remain occult until locally advanced and given the complex anatomy of the midface obtaining adequate margins during surgical resection is more difficult. It is these factors rather than the site of origin that affect prognosis. Cervical metastases in maxillary sinus squamous cell carcinoma: Despite typically presenting with advanced T-stage, clinical or radiographic nodal involvement at the time of presentation was uncommon in patients with maxillary sinus SCC. In our study post treatment failure more commonly occurred at the primary site than as a result of regional metastases.
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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.