Investigating RNF43 Mutations as Biomarkers for ctDNA Surveillance of Colorectal Cancer

Abstract

This project aimed to investigate the utility of RNF43 mutations for circulating tumour DNA (ctDNA) surveillance of colorectal cancer (CRC). CRC is the second deadliest cancer in New Zealand. CRC patients currently under-go 5 years of follow-up surveillance post-resection of primary tumours, requiring CT scans, colonoscopies, and CEA-tests to detect disease recurrences. These methods can be invasive, difficult to access, and inaccurate. Surveillance with ctDNA during and after treatment is a promising alternative to these current methods. ctDNA is DNA released into the bloodstream by dying tumour cells. It carries tumour-specific information such as somatic mutations and methylation changes that distinguish it from normal cell-free DNA in the bloodstream. ctDNA can be detected and quantified in patient plasma samples via assays for these tumour-specific alterations. ctDNA levels have been shown to correlate with tumour burden, disease progression, risk of recurrence, and real-time response to treatment. RNF43 is a tumour suppressor that plays an important role in regulating the Wnt signalling pathway that is frequently dysregulated in CRC. We investigated RNF43 mutations in a New Zealand CRC patient cohort to determine if they occur at a high enough frequency to justify their inclusion on custom targeted tumour sequencing panels being developed for the detection of personalised mutation markers for ctDNA surveillance of CRC. In this project, we designed a novel protocol for sequencing RNF43 that optimises time and cost efficiencies and yields highly accurate exome sequence data to identify true pathogenic mutations. Using this protocol, we identified pathogenic mutations in RNF43 in 3 of 24 (~13%) primary tumour samples. This fairly high mutation rate and the likely founder status of the pathogenic RNF43 mutations justifies the inclusion of the RNF43 gene in targeted sequencing panels for the routine detection and quantification of ctDNA markers in CRC.