The presence of epithelial dysplastic areas in potentially malignant oral lesions (PMOLs) is generally accepted as one of the most reliable predictors of malignant development. However, histopathologic diagnosis is subjective and lacks sensitivity. Therefore many attempts have been made to identify objective molecular biomarkers to identify, classify and prognosticate oral epithelial dysplasia (OED). This thesis endeavoured to discover new biomarkers for OED and oral squamous cell carcinoma (OSCC). In chapter 3 of this thesis, we reviewed microarray studies which analysed PMOLs and identified a set of commonly dysregulated genes and selected 5 genes for further analysis. The immunohistochemical expression pattern of calmodulin-like skin protein (CLSP), E74-like factor 3 (ELF3), interferon-induced protein 44 (IFI44), ubiquitin specific peptidase 18 (USP18), and chemokine-CXC-motif ligand 13 (CXCL13) was investigated in 18 normal oral mucosa (NOM), 27 mild dysplasia (MD), 13 moderate to severe dysplasia (SD) and 49 OSCC samples. Our results showed a gradual decrease of CLSP and ELF3 expression from NOM and MD to SD and OSCC. We also noted a gradual increase in staining intensity for IFI44 from NOM to MD to SD with peak expression in OSCC. The gradual decrease of CLSP and ELF3 expression may be explained by the fact that CLSP and ELF3 have a role in late keratinocyte differentiation and it is expected to see a lesser degree of differentiation in OSCC. IFI44 which was found to be over-expressed in SD and OSCC in this study has been linked to metastasis and lymph node involvement in breast and colon cancer in other studies.
In our journey to identify novel biomarkers, we sought to investigate the expression of representative cancer stem cell markers in OED and OSCC. In Chapter 4, we evaluated the expression of aldehyde dehydrogenase isoform 1 (ALDH1), p75, CD44, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), and CD24 in 18 NOM, 27 MD, 13 SD, and 49 OSCC specimens. Our work presented novel evidence that the expression of ALDH1, LGR5, and CD24 increased in parallel with the severity of OED. Significantly higher staining of ALDH1, LGR5 and CD24 was noted in OSCC samples compared with samples of mild dysplasia (MD) and normal. Likewise there was a significant increase in CD44 in OSCC compared to NOM. These results suggest that the characteristic expression of these markers may be of diagnostic and prognostic value for OED and should be investigated further as markers of cancer progression.
To investigate the usefulness of our candidate markers for detection of OED and OSCC, we selected the most robust markers (ELF3 and CD24) for further investigation. In chapter 5, a new cohort of 278 samples was immunostained with ELF3 and CD24. These samples consisted of 90 NOM, 40 MD, 21 SD, and 127 OSCC. Using the cut-off scores generated from the training data (samples used in chapters 3 and 4), ELF3 and CD24 exhibited a reasonable performance for distinguishing OSCC from non-malignant tissues. Regarding OED detection, ELF3 staining showed low sensitivity and specificity in distinguishing dysplasia from normal tissue indicating that this marker is not helpful in differentiating dysplastic lesion. On the other hand, using CD24 staining showed a good overall result as the correct diagnosis for presence of dysplasia was determined in 68.8% of cases (sensitivity, 0.68; specificity, 0.8; and AUC, 0.74). This indicates that CD24 may have additional diagnostic value when combined with routine histology. In the current study, we observed that neither single markers nor combination of markers was perfect in determining SD.
In chapter 6, we intended to quantitate ALDH-, p75-, CD44, and CD24-positive subpopulation in a range of source tissue dysfunction ranging from normal (OKF6-TERT2), mild to moderate dysplasia (DOK), severe dysplasia (POE-9n) and OSCC (PE/CA PJ15). Similar to our IHC results in chapter 4, we found that ALDH activity was increased with increased disease severity of the source tissue for the cell lines from normal, through dysplasia to OSCC. Furthermore, we showed that dysplastic and OSCC derived cell lines expressed increased amounts of membrane CD24 compared to normal oral keratinocyte derived cell line.
The last part of this thesis involved exploring the functional role of LGR5 in oral carcinogenesis. We applied the siRNA technology to knockdown Lgr5 gene expression in 2 OSCC derived cell lines (SCC-9 and SCC-25). The main finding of this chapter was the suppression of SCC-9 cell growth after knocking down LGR5 which was reproducible by using another independent siRNA dublex. This indicates a possible role for LGR5 in oral cancer cell proliferation. Given the previous observation that LGR5 has a possible role in colon cancer metastasis, we hypothesised that LGR5 knock-down would negatively impact oral cancer cell adhesion, migration, and invasion. To investigate that we conducted adhesion, wound healing, trans-well migration, and invasion assays. Our results collectively suggested that LGR5 knock down does not significantly affect adhesion, migration, or invasion of SCC-9 and SCC-25 cell lines.