Jonathan Evans

Laparoscopic preperitoneal mesh repair using a novel self-adhesive mesh

Prosthetic mesh is now used routinely in inguinal hernia repairs, although its fixation is thought to be a potential cause of chronic groin pain. The Parietene ProGrip™ (TYCO Healthcare) mesh, which is semi-resorbable and incorporates self-fixing properties, has been shown to provide satisfactory repair in open surgery. We describe the use of this mesh in TAPP hernia repair, which has not previously been reported in the literature. A prospective study of 29 patients showed a mean operative time to be 47.6 min, with 96% of patients discharged home on the day of surgery or the day after. Visual analog pain scales (out of 10) reduced from 4 preoperatively to 0 at 6 months, and only 1 patient suffered a minor wound complication. The use of this mesh in transabdominal preperitoneal hernia repair is therefore feasible, safe, and may reduce postoperative pain.

From mice to men: Murine models of colorectal cancer for use in translational research.

Colorectal cancer (CRC) is the third most common carcinoma worldwide and despite advances in treatment, survival for patients with metastatic disease remains poor. With nearly 50% of patients developing metastases, in vivo investigation is essential to improve outcomes for these patients and numerous murine models of CRC have been developed to allow the study of chemoprevention and chemotherapy, in addition to improving our understanding of the pathogenesis of CRC. Selecting the most appropriate murine model for a specific application will maximize the conversion of potential therapies from the laboratory to clinical practice and requires an understanding of the various models available. This review will provide an overview of the murine models currently used in CRC research, discussing the limitations and merits of each and their most relevant application. It is aimed at the developing researcher, acting as a guide to prompt further reading in planning a specific study.

The use of Gore Bio-A in the management of the open abdomen.

Non-permanent, non-woven options for the closure of an open abdomen have previously been limited to biologics such as Permacol or Strattice. Gore Bio-A is constructed from biocompatible synthetic fibres, the use of which has only been described in the repair of inguinal hernia, hiatal hernia and fistula-in-ano. A 60-year-old male underwent emergency laparotomy, partial gastrectomy and formation of a feeding jejunostomy for a strangulated and perforated intrathoracic hiatus hernia. His abdominal wall subsequently dehisced for which he underwent laparostomy and subsequent early closure with a Gore Bio-A mesh, secured in an onlay manner with 2/0 vicryl. Functional and cosmetic outcomes were satisfactory and the patient was discharged home. The use of Gore Bio-A is a safe, feasible and cost effective alternative to traditional biologics for the closure of a laparostomy, deployment of which is safe within a contaminated field. Further prospective data is needed to clarify its role.

Specific mutations in KRAS codon 12 are associated with worse overall survival in patients with advanced and recurrent colorectal cancer

Background:Activating mutations in KRAS have been suggested as potential predictive and prognostic biomarkers. However, the prognostic impact of specific point mutations remains less clear. This study assessed the prognostic impact of specific KRAS mutations on survival for patients with colorectal cancer.Methods:Retrospective review of patients KRAS typed for advanced and recurrent colorectal cancer between 2010 and 2015 in a UK Cancer Network.Results:We evaluated the impact of KRAS genotype in 392 patients. Mutated KRAS was detected in 42.9% of tumours. KRAS mutations were more common in moderate vs well-differentiated tumours. On multivariate analysis, primary tumour T stage (HR 2.77 (1.54–4.98), P=0.001), N stage (HR 1.51 (1.01–2.26), P=0.04), curative intent surgery (HR 0.51 (0.34–0.76), P=0.001), tumour grade (HR 0.44 (0.30–0.65), P=0.001) and KRAS mutation (1.54 (1.23–2.12), P=0.005) were all predictive of overall survival. Patients with KRAS codon 12 mutations had worse overall survival (HR 1.76 (95% CI 1.27–2.43), P=0.001). Among the five most common codon 12 mutations, only p.G12C (HR 2.21 (1.15–4.25), P=0.01) and p.G12V (HR 1.69 (1.08–2.62), P=0.02) were predictive of overall survival.Conclusions:For patients with colorectal cancer, p.G12C and p.G12V mutations in codon 12 were independently associated with worse overall survival after diagnosis.

Proteomic analysis to identify biomarkers in the primary tumour that predict response to neoadjuvant chemotherapy in liver metastases

BACKGROUNDnColorectal cancer is the fourth commonest cancer in the UK, and the second commonest cause of cancer-related death. A knowledge of the biological phenotype of colorectal liver metastases would be invaluable to inform clinical decision making; however, deriving this information from the metastatic lesions is not feasible until after resection. We aimed to use proteomic analysis to identify biomarkers in the primary tumour that predict response to neoadjuvant chemotherapy in liver metastases.nnnMETHODSnFresh tissue from both primary colorectal tumour and liver metastases from 17 patients was subjected to proteomic analysis using isobaric tagging for relative quantification. Data were analysed with Protein Pilot (Ab Sciex, Framingham, MA, USA), with stratification of patients into those showing low or high response to chemotherapy permitting the identification of potential predictive biomarkers. These markers were subsequently validated by immunohistochemistry on a tissue microarray of 63 patients.nnnFINDINGSnWe identified 5768 discrete proteins. Five of them predicted histopathological response to fluorouracil-based chemotherapy regimens, of which the FAD binding protein NQO1 was subsequently validated by immunohistochemistry. When compared with the chemotherapeutic agent alone, knockdown of the corresponding gene with small interfering RNA decreased cell viability when co-incubated with fluorouracil (77·1% vs 46·6%, p=0·037) and irinotecan (41·7% vs 24·4%, p=0·006). Similar results were also seen after inhibition of protein activity by pretreating cells with dicoumarol.nnnINTERPRETATIONnThese results show that proteomic sequencing of matched metastatic colorectal cancer samples is feasible, with high protein coverage. The high degree of similarity between the primary and secondary proteomes suggests that primary tissue is predictive of the metastatic phenotype. NQO1 expression in the primary tumour predicts response to neoadjuvant chemotherapy in the liver metastases, and inhibition of this protein at both genetic and functional levels improves chemosensitivity.nnnFUNDINGnCancer Research UK.

The Nrf2 inhibitor brusatol is a potent antitumour agent in an orthotopic mouse model of colorectal cancer

Nrf2 is a transcription factor that regulates cellular stress response and irinotecan-metabolising pathways. Its aberrant activity has been reported in a number of cancers, although relatively few studies have explored a role for Nrf2 in colorectal cancer (CRC). This study assessed the expression of Nrf2 in patient CRC tissues and explored the effect of Nrf2 modulation alone, or in combination with irinotecan, in human (HCT116) and murine (CT26) cell lines in vitro and in an orthotopic syngeneic mouse model utilising bioluminescent imaging. Using a tissue microarray, Nrf2 was found to be overexpressed (p<0.01) in primary CRC and metastatic tissue relative to normal colon, with a positive correlation between Nrf2 expression in matched primary and metastatic samples. In vitro experiments in CRC cell lines revealed that Nrf2 siRNA and brusatol, which is known to inhibit Nrf2, decreased viability and sensitised cells to irinotecan toxicity. Furthermore, brusatol effectively abrogated CRC tumour growth in subcutaneously and orthotopically-allografted mice, resulting in an average 8-fold reduction in luminescence at the study end-point (p=0.02). Our results highlight Nrf2 as a promising drug target in the treatment of CRC.

Exome sequencing of synchronously resected primary colorectal tumours and colorectal liver metastases to inform oncosurgical management

BACKGROUNDnNext generation sequencing technology has facilitated mapping of the colorectal cancer genotype and furthered our understanding of metastogenesis. The aim of this study was to investigate for conserved and different mutations in the exomes of synchronously resected primary colorectal tumour and liver metastases. This information could potentially be utilised to guide the treatment of advanced disease with the help of biological information from the primary tumour.nnnMETHODSnWe performed exome sequencing of synchronously resected primary colorectal cancer and colorectal liver metastases as well as normal colonic mucosa and liver parenchyma, from four patients who had received neo-adjuvant chemotherapy, at a depth of 50X using the Ion Proton platform. Raw data was mapped to the reference genome prior to variant calling, annotation and downstream analysis.nnnRESULTSnExome sequencing identified 585 non-synonymous missense single nucleotide variants (SNVs), of which 215 (36.8%) were unique to the primary tumour, 226 (38.6%) unique to the metastasis and 81 (13.8%) present in patient matched pairs. SNVs identified in the ErbB pathway appear to be concordant between primary and metastatic tumours.nnnCONCLUSIONnOnly 13.8% of the metastatic exome can be predicted by the genotype of the primary tumour. We have demonstrated concordance of a number of SNVs in the ErbB pathway, which may inform selection of therapeutic agents in advanced colorectal cancer.

Evaluation of NQO1 as a predictive biomarker and therapeutic target in metastatic colorectal cancer

Abstract Background A knowledge of the biology of colorectal liver metastases would be invaluable to inform clinical decision making; however, deriving this information from metastatic lesions is not feasible until after resection. We aimed to use proteomic analysis to establish the degree of biological similarity across disease sites and identify and validate biomarkers in the primary tumour that predict response to neoadjuvant chemotherapy in liver metastases. Methods Tissue from primary colorectal tumour and liver metastases (n=16) were subjected to proteomic analysis with isobaric tagging for relative quantification. Data were analysed with ProteinPilot software (Ab Sciex, Framingham, MA USA) with stratification of patients into low or high response to chemotherapy. These biomarkers were investigated by immunohistochemistry on a tissue microarray of 56 patients. Their therapeutic potential was investigated by dosing of SW480 cells with irinotecan or fluorouracil with or without inhibition by small interfering RNA (siRNA) or a competitive inhibitor (dicoumarol). Findings We identified 5766 discrete proteins, of which 2·54% were differentially expressed between primary and metastatic tumours. There were 170 potential response biomarkers in the primary tumour and 27 in the metastases. Lambda-crystallin homolog and NQO1 were common to both tissue types and showed consistent dysregulation. Immunostaining of NQO1 in metastatic tissue was lower in patients responding than in those not responding to chemotherapy (p=0·041), with a significant correlation between primary and metastatic disease sites (r=0·44, p=0·001). Knockdown of NQO1 with siRNA followed by treatment with irinotecan or fluorouracil reduced the IC50 from 100·1 to 49·8 μM and from 200·1 to 25·0 μM, respectively. Treatment of cells with dicoumarol before incubation in irinotecan or fluorouracil reduced the IC50 from 100·0 μM to 50·0 μM and from 183·7 μM to 49·9 μM, respectively. Interpretation We show that proteomic sequencing of matched metastatic colorectal cancer samples is feasible with high coverage. The high degree of similarity between the primary and secondary tumours suggests that the primary tissue is predictive of the metastatic phenotype. NQO1 expression in the primary tumour predicts response to neoadjuvant chemotherapy in liver metastases, and inhibition of this protein at both genetic and functional levels improves chemosensitivity. Funding Cancer Research UK.

PTH-272 Modulation of NRF2 alters the responsiveness of colorectal cancer cells to irinotecan

Introduction Chemotherapy is essential to improve outcomes in colorectal cancer (CRC). Irinotecan is a pro-drug commonly used in CRC which requires conversion to the active metabolite Sn38. The cytoprotective protein NRF2 has been associated with chemo-resistance and a worse prognosis in a number of cancers.1It also regulates the activity of enzymes (including CES1 and UGT1A1) that coordinate irinotecan metabolism and efflux. 2NRF2 remains bound to the regulatory protein KEAP1 in its quiescent state. The aim of this study is to establish the value of modulating NRF2 as a novel strategy for enhancing the efficacy of irinotecan-based chemotherapy either through increased conversion of irinotecan to Sn38 or by sensitisation of CRC cells to its effect. Method NRF2 was modulated in the human CRC cell line HCT116 using small inhibitory RNA (siRNA) targeting NRF2and KEAP1 or the chemical inhibitor brusatol and inducer CDDO-Me. Cells were then exposed to irinotecan over a range of concentrations for 48 h. Cell viability was measured by MTS assay and expressed as a percentage of control for the calculation of IC50values. GraphPad Prism 6®was used for all statistical analysis and the generation of dose-response curves. Results Successful NRF2 modulation was confirmed by western blotting for Nrf2 and downstream targets NQO1 and HO-1. The IC50 value of irinotecan in untreated control cells was 90µM. Induction of NRF2 by KEAP1siRNA (IC50 220µM) or CDDO-Me (IC50 325µM) significantly reduced irinotecan cytotoxicity (p < 0.0001). In contrast, inhibition of NRF2 by NRF2siRNA (IC50 11µM) or brusatol (IC50 55µM) had the opposite effect (p < 0.0001). Conclusion Inhibition of NRF2 sensitises CRC cells to irinotecan cytotoxicity and may allow improved response to treatment, or permit lower doses with fewer side effects. We are currently validating these findings in vivo and examining the contribution of processes that mediate the disposition of irinotecan. The availability of NRF2 inhibiting and inducing compounds may allow for successful modulation in the clinical setting. Disclosure of interest J. Evans Grant/ Research Support from: Cancer Research UK, B. Winiarski: None Declared, P. Sutton: None Declared, D. Palmer: None Declared, N. Kitteringham: None Declared. References Kawasaki Y, Ishigami S, Arigami T, Uenosono Y, Yanagita S, Uchikado Y, et al. Clinicopathological significance of nuclear factor (erythroid-2)-related factor 2 (Nrf2) expression in gastric cancer. BMC Cancer 2015;15(1):5 Wang M, Zhu JY, Chen S, Qing Y, Wu D, Lin YM, et al. Effects of co-treatment with sulforaphane and autophagy modulators on uridine 5’-diphospho-glucuronosyltransferase 1A isoforms and cytochrome P450 3A4 expression in Caco-2 human colon cancer cells. Oncol Lett. 2014;8(6):2407–16

PWE-305 Nqo1 is a predictive biomarker and therapaeutic target in metastatic colorectal cancer

Introduction A knowledge of the biological phenotype of colorectal liver metastases would be invaluable to inform clinical decision making; however, deriving this information from the metastatic lesions is not feasible until after resection. We aimed to use proteomic analysis to identify biomarkers in the primary tumour which predict response to neoadjuvant chemotherapy in liver metastases. Method Fresh tissue from both primary colorectal tumour and liver metastases from 17 patients was subjected to proteomic analysis using isobaric tagging for relative quantification, with stratification of patients into those showing low or high response to chemotherapy permitting the identification of potential predictive biomarkers. These markers were subsequently validated by immunohistochemistry on a tissue microarray of 57 patients and further investigated in an in vitro model. Results We identified 5768 discrete proteins, 5 of which predicted histopathological response to fluorouracil-based chemotherapy regimens. Immunostaining of NQO1 in the metastases was lower in those responding to chemotherapy (p = 0.041), with a significant correlation between primary and metastatic disease sites (r = 0.44, p = 0.001). Knockdown of NQO1 with small interfering RNA followed by treatment with irinotecan and 5FU reduced the IC50 from 100.1µM to 49.8µM and from 200.1µM to 25.0µM respectively. Pre-treating cells with dicoumarol (a known competitive inhibitor) prior to incubation in irinotecan and 5FU reduced the IC50 from 100.0µM to 50.0µM and from 183.7µM to 49.9µM respectively. Conclusion These results show that proteomic sequencing of matched metastatic colorectal cancer samples is feasible, with high protein coverage. The high degree of similarity between the primary and secondary proteomes suggests that primary tissue is predictive of the metastatic phenotype. NQO1 expression in the primary tumour predicts response to neoadjuvant chemotherapy in the liver metastases, and inhibition of this protein at both genetic and functional levels improves chemosensitivity. Further validation in an in vivo model is planned. Disclosure of interest P. Sutton Grant/ Research Support from: Cancer Research UK, J. Evans: None Declared, R. Jones: None Declared, C. Goldring: None Declared, D. Palmer: None Declared, N. Kitteringham: None Declared, H. Malik: None Declared, D. Vimalachandran: None Declared.