Prognostic role of tumour associated trypsin inhibitor in colorectal cancer patients

Sammanfattning: Colorectal cancer (CRC) is one of the most common forms of human cancer worldwide with approximately 1 million new cases detected every year. CEA is currently the only accepted CRC marker incorporated into clinical practice, where it is used for early detection of metastasis in follow-up of patients having stage II and III disease, and for monitoring response to adjuvant treatment. However there are no reliable prognostic and response predictive biomarkers for CRC. Tumour-associated trypsin inhibitor (TATI) is a form of antitrypsin, a 6kDa enzyme that inhibits trypsin with great affinity and TATI has previously been found to be of prognostic importance in mucinous ovarian cancer and other cancer forms. TATI is expressed in normal colonic mucosa, where its main function is to act as an antitrypsin, protecting the tissue from proteolysis. TATI has also been shown to promote migration and invasion in colorectal cancer cell lines in vitro. Moreover, TATI has a similar molecular structure to epidermal growth factor (EGF) and has been found to bind to EGFR and promote malignant behaviour in various cell line models. The prognostic significance of TATI in CRC patients had however not been reported and the main aim of the present investigation was therefore to examine levels of TATI in tumour tissue and serum samples from three independent CRC patient cohorts, with particular reference to their association with clinical outcome. In addition, we assessed the effect of neoadjuvant radiation therapy (RT) on TATI levels in tissue and serum of rectal cancer patients, and whether expression of epidermal growth factor receptor (EGFR) and markers of downstream signalling have a modifying effect on the prognostic value of TATI. In paper I, we investigated TATI expression by immunohistochemistry (IHC) in CRC tumours (t-TATI) in two cohorts adding up to 424 patients. High TATI expression was found to be significantly associated to short overall survival (OS), and disease free survival (DFS), as well as an increased risk of liver metastasis. In paper II, we analysed the prognostic value of TATI in serum (s-TATI) in one of the cohorts used in Paper I (n = 334). While no significant correlation was found between s-TATI and t-TATI concentrations, the prognostic value of s-TATI was more evident than for t-TATI, the former being a strong independent predictor of an impaired OS, DFS and time to recurrence (TTR), suggesting that elevated serum concentrations are not a result of TATI production by the tumour. Further results from paper II revealed that s-TATI concentrations were lower in rectal cancer patients, the majority of whom had received neoadjuvant radiotherapy (RT). In paper III, we therefore ventured to investigate whether neoadjuvant RT affects TATI concentrations in tissue and serum. To this end, we analysed serum and tissue samples collected at multiple timepoints from 53 patients with rectal cancer included in a in a case-control study. The results revealed that t-TATI and s-TATI levels remained unaffected by RT. S-TATI was significantly associated with shorter OS at all timepoints, and t-TATI assessed in surgical specimens was also a factor of poor prognosis. In paper IV we investigated the associations of s-TATI and t-TATI to EGFR expression and downstream signalling proteins pSTAT3 and pERK1/2, as well as KRAS mutation status. Apart from a significant association between t-TATI expression and KRAS mutation status, there was no intercorrelation between t-TATI or s-TATI and the investigative markers. Expression of pERK1/2 and pSTAT3, and KRAS mutation status, was significantly associated with poor prognosis. The prognostic value of EGFR expression, being non-significant per se, was modified by s-TATI concentrations in that patients with EGFR positive tumours/ high s-TATI levels had a significantly shorter survival compared to patients with EGFR negative tumours/ low s-TATI levels.