Abstract
Endometrial cancer is a prevalent gynaecological malignancy, with rising incidence in Aotearoa New Zealand (NZ). Genomic advances revealed the importance of molecular profiling in endometrial cancer and investigating molecular features with prognostic capabilities. An example of this is CTNNB1, which encodes for β-catenin involved in the Wnt/β-catenin signalling pathway. Exon 3 CTNNB1 mutations are associated with an aggressive subset of endometrial cancer, commonly occurring in younger women with increased recurrence and worse patient outcomes. This project aimed to investigate the impact of CTNNB1 mutations in a NZ cohort using Sanger sequencing and assessing if β-catenin immunohistochemistry can be used as a surrogate marker for CTNNB1 mutations. A 3D organoid model was established from endometrial cancer tissue to investigate cancer recurrence associated with activating CTNNB1 mutations.
A cohort of 135 endometrial carcinoma samples were selected for Sanger sequencing from the University of Otago, Wellington Cancer Tissue biobank and underwent DNA extraction, PCR amplification for the exon 3 region of CTNNB1, Gel electrophoresis and Sanger sequencing. The sequenced electropherograms were analysed using Geneious Prime and mutations were compared to large-scale databases such as ClinVar and cBioPortal. A subset of matched samples from the sequencing cohort were selected for histological analysis. Immunohistochemistry was conducted for the β-catenin protein and scored based on cytoplasmic and nuclear expression. A 3D endometrioid, endometrial cancer organoid model was established using patient derived tissue. Downstream analysis of organoids involved measuring cell viability in response to Wnt modulators as a proxy for activating CTNNB1 mutations and RT-qPCRs to measure gene expression of targets associated with cancer recurrence.
CTNNB1 mutations were present in 11.6% of the cohort. They were significantly associated with younger patients (p = 0.0026) and were more common within Pacific peoples and Asian patients, being less common in NZ European patients. Cytoplasmic or nuclear staining of β-catenin did not associate with CTNNB1 mutations, nuclear staining of β-catenin resulted in a sensitivity of 0% and specificity of 83.3%. Organoids were successfully established and showed a similar trend of cell viability in presence of Wnt modulators. Both Wnt stimulator-RSPO1 and Wnt inhibitor-MSAB initially increased cell viability of organoids but over time cell viability for organoids in RSPO1 were significantly higher than MSAB. RTqPCRs of gene targets were unsuccessful.
This project confirmed the impact of CTNNB1 mutations affecting a subgroup of the NZ cohort, validating previous research in the literature and elucidating the role of CTNNB1 as a potential molecular marker. Findings suggest that β-catenin immunochemistry is not an ideal surrogate marker for CTNNB1 mutations and alternative methods should be investigated. These could include next-generation sequencing panels for key genes implicated in endometrial cancer or immunohistochemistry for other markers associated with CTNNB1 mutations. Patient-derived organoids show promise in measuring inter-patient variability in response to activating CTNNB1 mutations and could improve understanding of tumorigenesis-related molecular mechanisms. Future research in optimising endometrial cancer organoid culture and organoid RT-qPCR methodology would prove beneficial and could lead to more personalised treatment options for cancer research.