Evaluating the role of neurotensin and its receptors in colorectal cancers and polyps

Abstract

Importance Colorectal Cancer (CRC) accounts for 9% of cancer deaths globally. The clinical role of hormones is well established in some cancers but remains unclear in CRC. Neurotensin (NTS) and its receptors 1 and 3 (NTSR1 and NTSR3) are implicated in CRC carcinogenesis and growth. This thesis hypothesises that NTS and its receptors have the potential to be used as novel diagnostic and prognostic biomarkers for CRC. Such biomarkers could improve early detection and risk stratification of CRC. Furthermore, it could allow the personalisation of CRC treatment through novel therapeutic pathways. Objectives The up to date evidence for the role NTS plays in CRC is systematically reviewed. Two studies were then carried out to test the hypothesis. Firstly, the accuracy of plasma NTS concentration as a diagnostic biomarker for colorectal polyp and cancer is assessed. The second study characterised and quantified the protein expression of NTS, NTSR1 and NTSR3 in human CRC specimens. It then evaluated the association between NTS, NTSR1 and NTSR3 expression, histological grade of CRC and cancer survival. Design, Setting, and Participants Two separate multicentre prospective cohort studies were set up. The first study recruited 180 patients to evaluate the diagnostic accuracy of plasma NTS concentration for colorectal polyps and cancers in patients with high-risk symptoms. Plasma samples were collected from the participants who then underwent diagnostic colonoscopy or computerised tomographic virtual colonography. The plasma concentration of NTS was quantified using enzyme-linked immunoassay. The diagnostic accuracy of plasma NTS was assessed using receiver operator characteristics curve analysis. The finding of the prospective study was then validated using 100 plasma samples stored in a tissue bank. The second study recruited 64 patients with CRC who underwent surgical resection with curative intent. The protein expression of NTS, NTSR1 and NTSR3 in the cancer resection specimens and surrounding normal epithelium was visualised using immunohistochemical staining. The intensity and proportion of expression of all three markers were quantified using the hybrid scoring (H-score) system. The relationship between the expression of these proteins and histopathological, clinical, and survival outcomes was evaluated. Results In the first study, of the 180 participants, 165 were eligible. Of these, 46 had colorectal polyps or cancer. Significantly higher plasma NTS was found in patients with colorectal polyps or cancers compared to those without (603.6 pg/ml vs 407.2 pg/ml, p < 0.01). Loge (plasma NTS concentration) was a significant independent risk factor for the presence of colorectal polyps or cancers (odds ratio, 2.73; 95% CI, 1.33–5.59, p < 0.01). Plasma NTS had an optimal sensitivity of 70.8% and specificity of 59.5% for the diagnosis of colorectal polyps and cancers. Similar diagnostic accuracy was obtained in the validation group. In the second study, of the 64 patients enrolled, 58 participants’ specimen blocks were immunostained. There was significantly higher expression of NTS in CRC of higher T stages (p<0.01), N stages (p=0.03), and AJCC clinical stages (p=0.04). There was significantly higher expression of NTS in cancer tissue compared to surrounding normal epithelium (median H-score 163.5 vs 97.3, p<0.01). There was significantly shorter disease-free survival (DFS) in individuals with CRC which expressed high levels of NTS compared to low levels of NTS (32.8 months 95% CI: 26.8 – 38.8 months vs 41.0 months 95% CI: 37.7–44.4 months respectively, p=0.01). Above median NTS expression in cancer tissue was a significant risk factor for disease recurrence (hazard ratio 5.60, 95% CI 1.22 – 25.6, p=0.03, Cox proportional regression). Conclusions and Relevance Plasma NTS has the potential to be a non-invasive biomarker for colorectal neoplasia. It appears to have better accuracy than existing blood markers and is unique in being able to also detect precancerous polyps. Furthermore, high tissue expression of NTS appears to be associated with more advanced CRC and shorter DFS. NTS expression level can potentially aid postoperative selection for conventional adjuvant therapy and identify individuals for novel therapies targeting the neurotensinergic pathways. These findings support the overarching hypothesis of this work; NTS has the potential to be both a diagnostic and prognostic marker of CRC.