Background: Medulloblastoma accounts for 18% of all paediatric brain cancers with between 50 and 100 diagnoses in the UK each year. There are four molecular subgroups with distinct clinical characteristics, cellular origins and identifying mutations. The four molecular subgroups are WNT, SHH, Group 3 and Group 4. The same treatment is given to all patients regardless of subgroup, and the majority of patients over three years receive craniospinal irradiation. The WNT and SHH subgroups are named after the overactive signalling pathways found in these tumours; these signalling pathways are involved in proliferation and response to radiation. The underlying biology of the molecular subgroups could contribute to differences in radioresponsiveness through differences in intrinsic radiosensitivity, proliferative capacity and extent of tumour hypoxia. An understanding of subgroup-specific differences in radioresponsiveness would guide the personalisation of treatment based on a tumourâs molecular subgroups. Aims and Objectives: The aim of this project was to identify factors that might affect differences in the radioresponsiveness of the medulloblastoma subgroups. The specific objectives were to investigate differences in radiosensitivity, proliferation, tumour hypoxia and cell migration between the four molecular subgroups. Methods: A panel of medulloblastoma cell lines representing the SHH and Group 3 subgroups was used in two radiosensitivity assays (a clonogenic and high-throughput assay) and a migration (gap closure) assay. The clonogenic assay was carried out with the adherent cell lines under normoxic and hypoxic (0.1% oxygen) conditions. The high-throughput assay work was carried out under normoxic conditions only. Radiation survival curves were fitted using a linear quadratic model. In silico analyses of publically available clinical cohorts were used to investigate a tumour radiosensitivity signature. Publically available data for clinical cohorts were also used to investigate proliferation measured as the mRNA expression of Ki67 and PCNA, and using a published gene signature. Tumour hypoxia was assessed using CAIX and GLUT1 mRNA expression data and published gene signatures. The gap closure assay was used with the adherent cell lines using barrier (Ibidi insert) and scratch (EssenBio WoundMaker) methods of gap creation. Cells were irradiated immediately, 24 h or 7 days prior to gap creation. Gap closure was monitored using time-lapse microscopy over 24 hours and image analysis carried out using software written for this thesis. Results: The in vitro radiosensitivity assays showed SHH cell lines to be more radiosensitive than Group 3 cell lines. The in silico work confirmed this, reporting SHH as the most radiosensitive subgroup, followed by the WNT, Group 3 and Group 4 subgroups. Clinical cohort data showed proliferation was not prognostic in medulloblastoma, and no significant differences in proliferation between the molecular subgroups. The hypoxia biomarkers and gene expression signatures were also not prognostic in medulloblastoma. However, the molecular subgroups were associated with different patterns of hypoxia-associated gene expression. The least hypoxic subgroup was WNT followed by the SHH, Group 4 and Group 3 subgroups. The SHH subgroup cell lines were more migratory within the gap closure assay than the Group 3 cell line. Irradiation produced cell line-specific alterations in gap closure rate but no subgroup-specific alteration of migration following radiation treatment was observed. Discussion: Subgroup-specific differences in intrinsic radiosensitivity and level of hypoxia are found within medulloblastoma and may contribute to subgroup-specific differences in radioresponsiveness and clinical outcomes. Further clarification of the role of hypoxia in medulloblastoma is required to confirm this relationship.
Targeting medulloblastoma: Does subgroup-specific radioresponsiveness affect outcomes?
Morris, R. (Author). 31 Dec 2021
Student thesis: Phd