Discovery and validation of podocalyxin-like protein as a prognostic biomarker in colorectal cancer

Abstract: Colorectal cancer (CRC) is the third most common cancer worldwide with more than 700,000 deaths every year. Prognosis mostly depends on disease stage at diagnosis, however, outcome may vary considerably even within the same stage. Thus, there is a great need for new prognostic biomarkers to better identify patients with a high risk of developing metastases and select right patients for adjuvant treatment. Podocalyxin-like protein (PODXL) has been associated with an aggressive tumour phenotype and adverse outcome in different types of cancer. The aim of this thesis was to investigate the prognostic and predictive significance of PODXL in CRC. In addition, the associations between PODXL and other biomarkers in CRC, including EGFR and BRAF, were examined. Expression of PODXL and EGFR was examined by immunohistochemistry on tissue microarrays containing tumours from three independent CRC patient cohorts (n=557, n=270 and n=320 respectively). BRAF mutational status was assessed by pyrosequencing, and quantitative polymerase chain reaction was used to compare mRNA levels and PODXL protein expression in 62 tumours. Further, concordance between PODXL expression in primary tumours and lymph node metastases, and in tumour samples pre- and postirradiation was examined in a subset of tumours (n=31 and n=16 respectively). Finally, western blot was used to analyse expression of PODXL and EGFR in six CRC cell lines. High PODXL expression was associated with unfavourable clinicopathological parameters and was found to be an independent factor of poor prognosis in CRC. Furthermore, CRC stage III patients with tumours displaying high PODXL expression had a significant benefit from adjuvant chemotherapy, whereas patients with tumours displaying low PODXL expression did not. We found no correlation between mRNA levels and protein expression of PODXL, and the expression of PODXL did not differ between primary tumours and lymph node metastases, nor was it altered by neoadjuvant radiotherapy. Furthermore, PODXL expression was found to correlate with EGFR expression and BRAF mutation, and in vitro studies showed that PODXL and EGFR were expressed in a uniform way in the cell lines. The highest risk of death within 5 years was observed in patients with high expression of both EGFR and PODXL. In conclusion, the results from this thesis demonstrate for the first time that PODXL is an independent factor of poor prognosis in CRC, and a potential predictive marker for stratifying patients for adjuvant chemotherapy.