Characterization of oral leukoplakia for the prediction of malignant transformation

In Chapter 2 in total 170 patients were studied to investigate the relation between a set of clinical variables and risk for MT of OL, such as age, gender, smoking habits, clinical presentation, lesion subsite and size and treatment. Only presence of OL at specific high risk subsites, tongue or floor of mouth, appeared as a clinical predictor of MT. In addition, it was shown that the annual MT rate of OL was 4.9% and this remained more or less consistent for the entire follow-up period. Recently, differentiated dysplasia was identified as a new pattern of dysplasia in vulvar epithelial lesions, but this pattern was not yet recognized in OL. As a proof of concept, we studied in Chapter 3 the OL lesions of 84 patients and scored all lesions for presence and grade of dysplasia. Time to MT was significantly longer for lesions with architectural dysplasia compared to lesions with classic epithelial dysplasia. When classic epi-thelial dysplasia and architectural dysplasia were combined, only two out of out of 30 lesions without any pattern of dysplasia transformed to OSCC. These results were subsequently corroborated in a much larger cohort of 176 patients in Chapter 4. In addition, we found a moderate inter-observer agreement for the diag-nosis of architectural dysplasia in OL lesions. We hypothesized that by combining morphological changes with genetic aberrations it would be possible to identify a group of patients with a very high risk for MT. In Chapter 5 we included 89 patients and used low coverage whole genome sequencing and targeted mutational sequencing to identify CNAs and mutations in the 12 most important HNSCC cancer genes, respectively. Most OL lesions harbored at least one genetic event, most often gains of chromosome regions 8q24 and 20p11, loss of 13q12 and mutations in TP53, FAT1 and NOTCH1. Together with presence of dysplasia we were able to identify three groups of patients with a distinct risk for MT of OL. In Chapter 6 we studied OL lesions of 21 patients that eventually developed into an OSCC. For each patient the first OL biopsy, the tumor biopsy and all available intermediate biopsies were collected, and DNA se-quencing for CNAs and mutations was performed. The phylogenetic history of all lesions was reconstructed, taking the applied interventions into account. We identified three different routes of progression. In addition to providing new insights in progression of OL lesions, it was possible to use the obtained genetic profiles and risk models to predict MT of OL in patients in a clinical setting. In Chapter 7 we describe the development of a non-invasive genetic assay on brushed cells to detect presence of genetically altered fields, even when not visible to the clinician. In Chapter 8 a well-established method for the generation of cell lines from oral biopsies was translated to the tumor-adjacent and OL keratinocyte setting, and this method was further improved by genomic engi-neering. The biopsies were cultured and genetically characterized. CRISPR/Cas9 was employed to introduce mutations in TP53 and CDKN2A which was combined with overexpression of TERT to improve success rates and to generate immortalized cell lines. Finally, a subset of cultures was used to assess the efficacy of a set of small-molecule inhibitors. To conclude, this thesis presents a comprehensive overview of the clinical, histomorphological and genetic background and evolution of OL. By combining these aspects into one model it was possible to accurately predict risk for MT of OL to OSCC. In vitro cell models were generated from patient tissue initiating the first steps required for the development of new treatments to prevent MT of OL.