Abstract
Endometrial Cancer (EC) is the most common gynaecologic malignancy in the developed world. Historically, EC has largely affected post-menopausal people, however, in NZ, the incidence of early-stage Endometrioid Endometrial Cancer (EEC) and pre-cancer (Atypical Endometrial Hyperplasia (AEH)) is rising in young people. Conservative treatment options, including the Levonorgestrel intrauterine system (LNG-IUS) are becoming more common for people wishing to retain fertility options, or for whom surgery may be high risk due to co-morbidities. Despite the growing adoption of LNG- IUS treatment, there are challenges associated with the current care pathway in NZ, and histological response rates to LNG-IUS exhibit wide variability. Approximately one-third of tumours are resistant to LNG-IUS treatment and the underlying cellular resistance mechanism remains unknown. This underscores the need to deepen the understanding of LNG-resistance and develop new biomarkers to predict and monitor response to LNG-IUS treatment.
Chapter Two investigated the LNG-IUS care pathway for AEH and EEC in NZ, focusing on incidence, clinical outcomes, and indications for LNG-IUS treatment at Te Whatu Ora (Health NZ) – Capital Coast and Hutt Valley Hospitals. Demographics and treatment indications for 82 patients undergoing LNG-IUS treatment were analysed. Histological complete response rates were 76% (25/33) for AEH and 33% (7/21) for EEC. The findings were then aligned with the NZ Gynaecology Oncology Group (NZGCG) guidelines for LNG-IUS treatment of AEH and EEC in NZ to identify service development needs. These included improving endometrial sampling, additional weight support for people with high weight, and molecular classification of EEC. The study also proposed exploring biomarkers for predicting and monitoring LNG-IUS treatment response.
Exploration of predictive biomarkers for conservative management of AEH and EEC is limited and there are currently no clinically utilised biomarkers for predicting and monitoring response to the LNG-IUS. Chapter Three employed a discovery based approach using RNA sequencing to identify differentially expressed genes (DEGs) between two LNG-resistant and matched sensitive EEC cell lines. Functional relevance of the DEGs was explored through gene ontology analysis, gene set enrichment analysis, and protein-protein interaction networks. Protein analysis identified HUB genes linked to LNG resistance, and validation was performed on the top 15 DEGs and top 10 HUB genes in resistant and sensitive EEC cell lines. The study highlighted potential biomarkers (MAOA, MAOB, ALDH1A1, CXCL8, and CD80) implicated in drug resistance mechanisms, such as increased presence of cancer stem cells, immune dysregulation, and evasion of apoptosis, warranting further investigation in future studies in-vivo and in-vitro.
Chapter Four subsequently investigated the expression of the candidate biomarkers identified in Chapter Three. Eighteen people with AEH or EEC treated with the LNG-IUS at Te Whatu Ora – Capital Coast and Hutt Valley were recruited. Protein expression of ALDH1A1, CXCL8, KLF4 and MAOA was investigated in serum samples using enzyme- linked immunosorbent assay (ELISA). Serum ALDH1A1 was significantly upregulated in people showing histological non-response to the LNG-IUS, and was significantly upregulated in people with EEC compared to AEH. Protein expression of ALDH1A1, KLF4 and MAOA was also investigated in endometrial biopsy samples. ALDH1A1 protein expression was observed to be upregulated in people showing histological non- response to the LNG-IUS. Lastly, previously identified microRNA markers of LNG-IUS response were investigated in plasma-derived Extracellular Vesicles (EVs), with miR- 504-3p found to be significantly upregulated in people showing histological complete response to the LNG-IUS.
Finally, Chapter Five incorporated and built on the findings from Chapters Three and Four, investigating the role of cancer stem-like cells in EEC LNG resistance. Cancer stem- like cells were isolated from an EEC cell line using flow cytometry based on cell surface expression of CD133, a well-established marker of cancer stem cells. Isolated CD133+ve cells were shown to have upregulated mRNA and protein expression of CD133 and ALDH1A1, better sphere-forming ability and were more resistant to LNG treatments than CD133-ve EEC cells, highlighting the potential role of cancer stem cells in LNG resistance.
This thesis utilised a multidisciplinary approach, encompassing clinical review, bioinformatic analysis of genes related to LNG resistance, and patient biomarker investigation, to advance the understanding of LNG resistance in NZ. Overall, the findings of this thesis highlight: 1) the current LNG-IUS care pathway in NZ may benefit from improvements, 2) ALDH1A1 and miR-504-3p may serve as predictive biomarkers for LNG-IUS treatment and should be investigated further, and 3) LNG resistance may be a result of increased presence of cancer stem-like cells in the endometrium. These findings emphasise the importance of personalised approaches in the conservative treatment pathway of AEH and EEC, providing insights for future research and clinical applications.