Predicting and modifying response to neoadjuvant therapy in colorectal cancer

Sutton, Paul
Predicting and modifying response to neoadjuvant therapy in colorectal cancer. PhD thesis, University of Liverpool.

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Abstract Background Colorectal cancer is the fourth commonest cancer in the UK, and the second commonest cause of cancer-related death. A knowledge of the biology 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 and genomic analysis to establish the degree of biological similarity across disease sites and identify biomarkers in the primary tumour which predict response to neoadjuvant chemotherapy in liver metastases. An identical approach was also used to identify predictors of response to neoadjuvant chemoradiotherapy in rectal cancer. Methods Fresh tissue from both primary colorectal tumour and liver metastases from 16 patients was subjected to proteomic analysis using isobaric tagging for relative quantification (iTRAQ). 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 investigated by immunohistochemistry on a tissue microarray of 56 patients, in parallel with a series of in vitro studies to investigate the concordance between primary and metastatic tumours of those proteins relevant to the activation and metabolism of 5-FU, irinotecan and oxaliplatin. The therapeutic potential of the identified biomarkers was also investigated by dosing SW480 cells with irinotecan/5FU with or without inhibition (using siRNA or a known competitive inhibitor) of the proteins of interest. Four of the 16 patients studied were resected synchronously, and tissue from these was also used for exome sequencing using the Ion Proton platform. Diagnostic, post-treatment and resection biopsies from 8 patients with rectal cancer were again subjected to iTRAQ with stratification into low or high response to neoadjuvant chemoradiotherapy to investigate potential response biomarkers. 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. Two proteins were common to both tissue types and showed consistent dysregulation, including NQO1. Immunostaining of NQ01 in metastases revealed lower expression in patients 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 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 prior to incubation in irinotecan and 5FU reduced the IC50 from 100.0µM to 50.0µM and from 183.7µM to 49.9µM respectively. Exome sequencing identified 585 non-synonymous missense 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. Aberrations in the ErbB pathway were identified in paired samples (ratio 0.07, p=5.87x10-7), which were validated by Sanger sequencing along with a potential response biomarker in the CDAN1 gene. Changes to the phenotype of the rectal tumour with chemoradiotherapy were modest, although it is clear that base excision repair (and in particular PARP1) remains of interest, and that acid ceramidase is a potential response biomarker and novel radiosensitiser. Interpretation 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 the liver metastases, and inhibition of this protein at both genetic and functional levels improves chemosensitivity.

Item Type: Thesis (PhD)
Additional Information: Date: 2015-09-10 (completed)
Subjects: ?? Q1 ??
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Depositing User: Symplectic Admin
Date Deposited: 19 Jan 2016 14:56
Last Modified: 17 Dec 2022 01:27
DOI: 10.17638/02025228