Catherine Poh

Early detection and intervention of oral premalignant lesions (OPLs) are at the forefront of clinical endeavors for oral cancer control. Quantitative cytology (QC) and loss of heterozygosity (LOH) have shown to be promising prognostic biomarkers for identifying high-risk OPLs. The objectives of this thesis are to optimize the screening utility of the QC algorithm by assessing nuclear morphology and explore if QC or LOH may serve as predictive biomarkers in treatment management of OPLs. In this thesis, we first used 169 brushing samples from 156 patients to examine the comparability between the MoticEasyScan and ClearCyte and 105 samples from 100 patients to construct a morphometric-based QC-model for screening. Random Forest Models and Receiver Operating Characteristic curves were used to determine the cut-offs to identify abnormalities at cellular and case levels. To evaluate LOH and QC as biomarkers for predicting topical treatment response, a set of 28 OPLs from 28 patients were included for analysis. The Fisher’s exact test was used to assess the predictive value of LOH and QC. The results from the MoticEasyScan system are comparable to those from the ClearCyte for QC analysis. We have identified problems in the previous system with staining batch variations and normalization and both have significant impacts on photometric features used for previous software development. To overcome these problems, we developed a new QC model using morphometric only features. To develop a screening test, using 94 morphometric features with 65.7% of abnormal votes for abnormal cells and 2.7% abnormal cells for abnormal cases, the new model (RF94) yielded a 100% sensitivity in the Test set. To triage OPLs for treatment, the model (mRF10), using 10 morphometric features with 67.3% of abnormal votes for abnormal cells and 6.7% abnormal cells for abnormal cases, yielded in 87.5% specificity in the Test set. Using mRF10 model, no significant differences in proportions of the persistent and resolved groups in QC or LOH were identified at the pre-treatment timepoints. LOH may not be sensitive predictive markers for topical treatment of OPLs. In conclusion, QC models using morphometric-based features can potentially be a useful screening test.

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With 50% 5-year survival rates, oral cancer is often diagnosed at a late stage. Detection of lesions at earlier stages results in better prognosis. DNA-image cytometry (DNA-ICM) is able to detect gross alterations of cellular DNA-content representing aneuploidy, a biomarker of malignancy. Health-Canada-approved DNA-ICM system, ClearCyte (Perceptronix Medical Inc.) with a cytopathologist's review have demonstrated high sensitivity (89%) and specificity (97%) in identifying high-grade lesions. The purpose of this study was to test whether ClearCyte technology can provide a robust automated oral cancer screening method without cytopathologist’s input.A set of 218 oral brushing samples of oral cancer (n=96), severe dysplasia (n=20), reactive lesions (n=52), and normal samples (n=50) were retrieved from the bio-bank of the pan-Canadian surgical trial and community oral mucosal clinics. Cells from these liquid-based brushing samples were prepared using Cytospin4 (Thermo Scientific, Waltham, MA) and Feulgen-thionin stain reaction. Following ClearCyte scan, nuclear features were automatically calculated, and cells categorized into DNA-ploidy groups by the software: “diploid” (0.85≤DI1.15), “hyperdiploid” (1.15≤DI1.7), “tetraploid” (1.7≤DI2.3), and “aneuploid” (DI≥2.3). The samples were randomized into training and test sets (70:30) based on patient’s age, sex, tobacco use and lesion site risk. The training set (77 abnormal, 72 control samples) was used to plot the receiver operating characteristic curves to determine the best predictors of pathology and identify their thresholds for optimal sensitivity. Based on the training set data, a new algorithm, iClearCyte, was created and validated using the remaining samples in the test set (n=65), where sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the iClearCyte result were calculated. Four (1.8%) out of 218 cases were excluded from the analysis due to inadequate cellular counts (400 nuclei). The proposed iClearCyte algorithm (>1 “aneuploid” cell or ≥1.7% combined “hyperdiploid” with “tetraploid” group frequency) identified high-grade samples with the sensitivity, specificity, PPV and NPV of 100.0%, 86.7%, 89.7%, and 100.0%, respectively. In conclusion, iClearCyte test has potential to serve as robust non-invasive oral cancer screening tool, bridging geographic gap between communities and oral specialists, promoting early oral cancer detection, decreasing the number of unnecessary invasive biopsies, and guiding lesion management.

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Oropharyngeal squamous cell carcinoma (OpSCC) patients have improved survival when tested positive for high-risk human papillomavirus (HR-HPV). However, tissue assessment of HPV status is currently not standardized and additional factors may influence survival among HPV-positive patients. The main objectives were to evaluate HPV detection methods and to identify possible factors that impact survival of OpSCC patients in British Columbia.We retrospectively analyzed 972 primary OpSCC patients diagnosed between 2000-2008 and referred to the BC Cancer Agency for treatment with curative intents of radiotherapy with or without concurrent chemotherapy. Patient charts were reviewed and collected information for demographics, smoking history, clinical assessments, treatment received, and outcomes. We analyzed two cohorts of Study Cohort, 244 cases with enough formalin-fixed, paraffin-embedded (FFPE) tissues for experiment, and General Cohort, 728 cases without tissues available. Experimental procedures included in situ hybridization (ISH) to detect DNA and RNA HPV and immunohistochemistry (IHC) to detect p16, p53, the retinoblastoma protein (pRB), cyclin D1, and Ki67. We used polymerase chain reaction (PCR) to detect type-specific HPV from cases with enough FFPE tissues for DNA extraction (n=41). Cox proportional hazard (Cox-PH) and Kaplan-Meier (KM) survival analysis were conducted to identify potential clinical and biological factors impacting on 5-year overall survival (OS), disease-specific survival (DSS), and development of loco-regional recurrence (LRR). The incidence rates of males increased from 3.2 to 7.6 per 100,000 whereas females declined from 1.1 to 0.8 per 100,000. The Study Cohort was relatively representative of the General Cohort. The Study Cohort of patients classified as ever-smokers, had tumours staged at T3/4, and received radiotherapy only had poorer 5-year OS, DSS, and LRR (p0.05). HPV was detected in 77.6% of patients. Using PCR as standard, DNA/RNA ISH to detect HPV was more specific than IHC p16. Stratification of patients by HPV status showed that HPV-positive/p53-positive and HPV-negative/cyclin D1-positive patients had significantly poorer DSS (p=0.03) and 5-year OS (p=0.02), respectively. In conclusion, both HPV burden and its prognostic significance were found in BC. ISH assessment may be used to determine HPV status. IHC assessments of p53 or cyclin D1 status may be prognostic indicators to guide treatment.

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For patients of oral squamous cell carcinoma (OSCC), tumour spread to regional lymph nodes reduces survival by half. On the account of this widely demonstrated fact, prophylactic neck treatment has been advocated for clinically node negative (cN0) necks of which the risk of nodal disease is considerably high. However, there is a lack of sensitive and specific marker to determine such risk and benefits of prophylactic treatment await confirmation. The first part of this thesis presents a population-based retrospective review on OSCC in British Columbia. The incidence of regional failure (RF) in early-stage, cN0 patients was 28%, with median time of only 10 months after local excision. This group of patients needed to be identified and treated at earliest time possible. Tumour depth of invasion (DOI) was significantly associated with RF (P=0.01). However, it has low accuracy in predicting nodal disease with AUC of 63%. Moreover, assessment of performance for 4mm cut-off of DOI showed 55% sensitivity and 68% specificity. Furthermore, we demonstrated that using DOI as an indicator of neck treatment resulted in 25% under-treated occult metastasis and 55% over-treated necks. Thus, we concluded that, at least for BC population, conventional histological attributes of tumour cannot predict RF and we need a new marker for risk assessment. The second part presents a pilot study exploring a novel approach of risk assessment by utilizing Quantitative Tissue Pathology (QTP) on tumour nests. We were able to quantitate and evaluate 120 features describing nuclear phenotypes of tumour cell nuclei and tissue architectures of tumour nests. Compared to node-negative (N0) group, cell nuclei of the node positive (N+) group had higher fractions of heterochromatin regions. Also, the combination of two features, which describe chromatin condensation, from the outermost two layers of tumour nests had performance of AUC 94%, sensitivity of 100% and specificity of 75% in discriminating N0 and N+ group. QTP may be a potential proxy for predicting the metastatic risk of OSCC. Further investigation on potential biomarkers in risk assessment for nodal disease of early-stage OSCC patients is warranted to provide precision management to improve mortality and reduce morbidity.

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Low-income residents from the Vancouver’s Downtown Eastside (DTES) are known to be medically underserved, but little is known about their oral health (OH) status. The objectives of this study are to: 1) determine OH characteristics (clinical and subjective) of low-income adults in this community; 2) identify explanatory factors for their OH status.Material and Methods: Screening clinics were set up in the DTES. Eligibility criteria were adults 19 years of age or over and residence in the DTES for the preceding 3 months. Data were collected through questionnaires and clinical examinations.Results: Among the 356 screened participants, most were males, middle-aged, less educated, and living with low-income (≤$20,000/year). Alcohol and tobacco consumption was common. About 80% had dental coverage, mostly publicly funded benefits (94%). Fifty (14%) participants were edentulous. Dentate participants (n=306), on average, had 3.8 decayed (Dt), 8.6 missing (Mt), 4.9 filled (Ft), 17.2 DMF teeth, and a care index (CI) of 41.5%. Many (86%) perceived a dental need. Although more participants with dental insurance had a dental visit within the past 12 months, there were no differences in OH indices between those with or without dental insurance. After adjusting gender and recruitment site, social (barriers to care & length of DTES residence), behavioural (brush/floss), and personal (HCV/methadone) factors were identifiable explanatory factors for the CI level. Conclusion: DTES low-income have poor oral health status and many perceive a need for dental care. We identified several factors that may affect their levels of dental care; however, this community may inherited more complicated social issues. Further investigations to truly understand their challenges and needs are required for a better oral health promotion in this community.

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With 50% 5-year survival rates, oral cancer is often diagnosed at late-stage. Detection of lesions at earlier stages results in better prognosis. Continued tobacco use after cancer treatment is a risk for recurrence. However, little is known of the process from lesion identification to diagnosis, impact of smoking behavioural changes and barriers for tobacco cessation in patients with a high-risk oral lesion (HRL) diagnosis.Two survey-type questionnaires were used to collect data on tobacco usage and patient experiences leading to HRL diagnosis. Patients attending the BC Cancer Agency diagnosed with HRLs within 12 months of interview were invited to participate in Study I. Patients who also smoked ≥100 cigarettes within 5 years of questionnaire were eligible for Study II.Among 150 Study I patients, 61% were self-identified (SIG) and 39% were professionally screened (PSG; 88% by dental professionals). PSG identified significantly more precancerous lesions compared with SIG (54% vs. 23%, P = 0.0003) and was more likely to screen ever smokers (P = 0.05). SIG showed significantly higher rates of time delay from detection to diagnosis ≥3 months, compared with PSG (58% vs. 36%, P = 0.007). Frequency of dental visits strongly correlated with identification of HRLs by healthcare professionals (P = 0.004). Common symptoms for SIG were pain (77%) and non-healing ulcers (62%), which prompted patients to seek healthcare, while 71% of PSG patients were asymptomatic. Surprisingly, almost half (47%) of patients were not aware of oral cancer.Of 58 Study II patients, main tobacco cessation barriers included stress (67%), smoking enjoyment (64%), and not being ready to quit (60%). Increased nicotine intake may be associated with increased difficulties with tobacco cessation. Knowledge of health risks from tobacco usage (71%) and tobacco cessation aids (43%) were main cessation facilitators. The HRL diagnosis was a pivotal factor for patients to stop or reduce cigarette consumption.Oral cancer screening by healthcare professionals, particularly dental professionals, can facilitate identification of earlier oral lesions at risk of cancer progression. Promotion of oral cancer awareness and tobacco cessation for patients and healthcare professionals may facilitate earlier diagnosis of oral lesions and prevent recurrent lesions.

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