EMT inducing transcription factor SIP1: a predictive biomarker of colorectal cancer survival and recurrence? A Patel, R Sreekumar, R Bhome, KA Moutasim, J Wigley, JN Primrose, E Sayan, A Mirnezami Introduction Colorectal carcinoma (CRC) is the second commonest cancers in the UK1. Metastases are the main reason for CRC related mortality2. Currently, there are no biomarkers of occult CRC metastasis. Epithelial mesenchymal transition (EMT) is a phylogenically conserved trans-differentiation program known to drive cancer spread by inducing formation of highly invasive and motile cancer cells3,4. Identifying and targeting this pool of tumour cells is a major challenge in cancer research. SIP1 belongs to the Zhfx1 family of EMT-inducing transcription factors5. SIP1 expression and functional consequences are yet to be studied in the setting of CRC. Thus we investigated SIP1 expression CRC patients and highlight its potential as a prognostic biomarker. FIGURE 1: Epithelial cells demonstrate increased motility and invasiveness when EMT is activated. Consequently these cells manifest a mesenchymal phenotype Impact of SIP1 expression on patient survival as emulated by Kaplan Meier method FIGURE 3: Impact of SIP1 expression on patient survival as emulated by KM method. (A) Overall survival rate (log rank 10.525, p=0.001) (B) DFS rate (log rank 17.968, p=0.001) (C) CSS rate (log rank12.237, p=0.0001) (D) Local recurrence (log rank 9.132, p=0.03) (E) Distal recurrence (log rank 10.527, p=0.01) Kaplan-Meier (survival analysis by log rank test, demonstrated SIP1 expression is associated with poor overall survival (log rank=10.525, P=0.001, Figure A), decreased DFS (log rank=17.968, P=0.001, Figure B) and CSS (log rank=12.237, P=0.0001, Figure C). Early mortality in SIP1 expressing patients was due to disease recurrence after surgical intervention (Figure D and E). Mean DFS was 51 months (95% CI: 44.515–58.438) in SIP1 positive patients, compared to 68 months (95% CI: 63.095–71.862) in SIP1 negative (log rank=10.525, P=0.001). Mean CSS for SIP1 positive vs SIP1 negative patients was 55 (95% CI: 48.400–61.764) vs. 68 months (95% CI: 63.848–72.575) respectively (log rank=12.237, P=0.0001). Independent prognostic marker for CSS and DFS TABLE 2: Multivariate Analysis by Cox proportional hazard regression Model SIP1 expression has been identified as am independent prognostic marker for both cohorts, predicting DFS and CSS Nuclear SIP1 positivity is an independent prognostic biomarker for CSS and DFS in CRC. Stratifying CRC patients according to SIP1 expression may have profound translational effect on patient prognosis and management in future years. To facilitate progression of the role of SIP1 as a biomarker to a prospective study, and potentially randomised clinical trial in the future, external validation using an independent patient cohort is now required. Lee J M et al. J Cell Biol 2006;172:973-981 Methods A prospective database of 228 patients undergoing surgical resection for CRC containing demographic, pathological and clinical outcomes was collated. Immunohistochemistry (IHC) was undertaken and scored by two independent pathologists. SIP1 positivity and its association with clinicopathological outcomes and survival were calculated using Chi-Squared and Kaplan–Meier methods respectively. Cox proportional hazard regression model was used to undertake multivariable analysis for Disease free (DFS) and Cancer specific survival (CSS). Results FIGURE 2: Differential SIP1 scoring – Present of nuclei SIP1 staining = positive. Absence of nuclei SIP1 staining = negative 46% (105/228) of our CRC cases were scored SIP1 positive by two independent pathologist. Patient Clinicopathological Outcomes TABLE 1: (Right) Clinicopathological correlation with SIP1 expression in CRC patients To clarify the correlation between SIP1 expression and carcinogenesis of CRC, the clinicopathological variables between SIP1 expression patterns were compared. Clinicopathological correlations by Pearson’s Chi2 and Fishers Exact Tests revealed positive expression of SIP1 statistically correlated with; >TNM stage (P=0.024) >Metastasis at diagnosis (P=0.023) >Lymph node involvement (P=0.015) >Tumour stage (P=0.036). a= Pearsons Chi2 Test b= Fishers Exact Test Characteristic Risk Ratio 95% confidence interval p- value   Cancer Specific Survival SIP1 positive 3.193 1.525 - 6.685 0.002 Disease Free Survival 3.464 1.773 – 6.764 0.0001 Characteristic Total no. SIP1 low expression SIP1 high expression p- value T Stage   I 0.036 a II 60 37 23 III 158 72 83 IV  0 TNM Score 44 20 24 0.024 b 106 58 48 68 21  Lymph Node Involvement Absent 144  83  61  0.015 a Present 27 41 77 Metastasis at diagnosis 196 107 89 0.023 a 16 4 12 Tumour Size (mm) <50 141 75 66 0.656a  >50  77  37  40 Conclusion and future work References 1. Boyle P, Langman J S, ABC of colorectal cancer: Epidemiology. BMJ. 2000;321(7264):805–808. 2. World Health Organization Cancer Incidence in Five Continents. Lyon: The World Health Organization and The International Agency for Research on Cancer; 2002. 3. Thiery JP and Sleernan JP, Complex networks orchestrate epithelial mesenchymal transition. Nature Reviews Molecular Cell Biology. 2006;(7):131-142 4. Thiery JP, Acloque H, Huang RY and Nieto MA, Epithelial mesenchymal transition in development and disease, Cell, 2009;(139):871-890 5. Kalluri R and Weinberg RA, The basics of Epithelial mesenchymal transition, Journal of Clinical Investigations, 2009;(119):1420-1428